Modified membrane type serine protease 1 (MTSP-1) polypeptides and methods of use

ABSTRACT

Provided are MTSP-1 polypeptides modified to have altered activity and/or specificity so that they cleave a complement protein, such as complement protein C3, to inhibit its activity and thereby inhibit complement activation. The modified MTSP-1 polypeptides that inhibit complement activation can be used for treatment of diseases and conditions in which complement activation plays a role. Such diseases and conditions include inflammatory diseases and diseases with an inflammatory component. Exemplary of these disorders are ischemic and reperfusion disorders, including myocardial infarction and stroke, sepsis, autoimmune diseases, ophthalmic disorders, such as diabetic retinopathies and macular degeneration, including age-related macular degeneration (AMD), and transplanted organ rejection, such as renal delayed graft function (DGF).

RELATED APPLICATIONS

This application is continuation of pending U.S. application Ser. No.17/066,398, filed Oct. 8, 2020, to Applicant Catalyst Biosciences, Inc.,and inventors Edwin L. Madison, Vanessa Soros, and Mikhail Popkov,entitled MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MTSP-1) POLYPEPTIDESAND METHODS OF USE, which is a divisional of U.S. application Ser. No.16/890,936, filed Jun. 2, 2020, now U.S. Pat. No. 10,954,501, toApplicant Catalyst Biosciences, Inc., and inventors Edwin L. Madison,Vanessa Soros, and Mikhail Popkov, entitled MODIFIED MEMBRANE TYPESERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES AND METHODS OF USE, which is adivisional of U.S. application Ser. No. 16/015,093, now U.S. Pat. No.10,781,435, filed Jun. 21, 2018, to Applicant Catalyst Biosciences,Inc., and inventors Edwin L. Madison, Vanessa Soros, and Mikhail Popkov,entitled MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MTSP-1) POLYPEPTIDESAND METHODS OF USE, which claims benefit of priority to U.S. provisionalapplication Ser. No. 62/523,735, filed Jun. 22, 2017, to Edwin L.Madison, Vanessa Soros, and Mikhail Popkov, entitled “MODIFIED MEMBRANETYPE SERINE PROTEASE 1 (MT-SP1) POLYPEPTIDES AND METHODS OF USE,” and toU.S. provisional application Ser. No. 62/664,051, filed Apr. 27, 2018,to Edwin L. Madison, Vanessa Soros, and Mikhail Popkov, entitled“MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES ANDMETHODS OF USE.”

Pending U.S. application Ser. No. 17/066,398, filed Oct. 8, 2020, alsois a divisional of U.S. application Ser. No. 16/015,093, now U.S. Pat.No. 10,781,435, filed Jun. 21, 2018, to Applicant Catalyst Biosciences,Inc., and inventors Edwin L. Madison, Vanessa Soros, and Mikhail Popkov,entitled MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MTSP-1) POLYPEPTIDESAND METHODS OF USE.

This application is a divisional of U.S. application Ser. No.16/890,936, filed Jun. 2, 2020, now U.S. Pat. No. 10,954,501, toApplicant Catalyst Biosciences, Inc., and inventors Edwin L. Madison,Vanessa Soros, and Mikhail Popkov, entitled MODIFIED MEMBRANE TYPESERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES AND METHODS OF USE.

This application also is a continuation of U.S. application Ser. No.16/015,093, now U.S. Pat. No. 10,781,435, filed Jun. 21, 2018, toApplicant Catalyst Biosciences, Inc., and inventors Edwin L. Madison,Vanessa Soros, and Mikhail Popkov, entitled MODIFIED MEMBRANE TYPESERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES AND METHODS OF USE.

Benefit of priority is claimed to U.S. provisional application Ser. No.62/523,735, filed Jun. 22, 2017, to Applicant Catalyst Biosciences,Inc., and to inventors Edwin L. Madison, Vanessa Soros, and MikhailPopkov, entitled “MODIFIED MEMBRANE TYPE SERINE PROTEASE 1 (MT-SP1)POLYPEPTIDES AND METHODS OF USE.” Benefit of priority also is claimed toU.S. provisional application Ser. No. 62/664,051, filed Apr. 27, 2018,to Applicant Catalyst Biosciences, Inc., and inventors Edwin L. Madison,Vanessa Soros, and Mikhail Popkov, entitled “MODIFIED MEMBRANE TYPESERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES AND METHODS OF USE.”

This application is related to International Patent Application SerialNo. PCT/US2018/038844, filed Jun. 21, 2018, entitled “MODIFIED MEMBRANETYPE SERINE PROTEASE 1 (MTSP-1) POLYPEPTIDES AND METHODS OF USE,” whichclaims priority to U.S. Provisional Application No. 62/523,735, and toU.S. Provisional Application No. 62/664,051.

The subject matter of each of these applications and patent isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Jul. 2, 2021 is 1.40 megabytes in size,and is titled 4939DSEQ000.txt.

FIELD OF THE INVENTION

Provided are modified MTSP-1 polypeptides that cleave a complementprotein, thereby, inhibiting complement activation. By virtue of thisinhibition the modified MTSP-1 polypeptides can be used for treatment ofdiseases and conditions mediated by complement or in which complementactivation plays a role. These diseases and conditions, include, but arenot limited to, ophthalmic indications, including macular degeneration,such as age-related macular degeneration (AMD), diabetic retinopathies,Stargardt disease, renal delayed graft function (DGF), ischemic andreperfusion disorders, including myocardial infarction and stroke,sepsis, autoimmune diseases, inflammatory diseases and diseases with aninflammatory component, Alzheimer's Disease and other neurodegenerativedisorders.

BACKGROUND

The complement (C) system is part of the immune system and plays a rolein eliminating invading pathogens and in initiating the inflammatoryresponse. The complement system of humans and other mammals involvesmore than 30 soluble and membrane-bound proteins that participate in anorderly sequence of reactions resulting in complement activation. Theblood complement system has a wide array of functions associated with abroad spectrum of host defense mechanisms including anti-microbial andanti-viral actions. Products derived from the activation of C componentsinclude the non-self-recognition molecules C3b, C4b and C5b, as well asthe anaphylatoxins C3a, C4a and C5a that influence a variety of cellularimmune responses. These anaphylatoxins also act as pro-inflammatoryagents.

The complement system is composed of an array of enzymes andnon-enzymatic proteins and receptors. Complement activation occurs byone of three primary modes known as the “classical” pathway, the“alternative” pathway and the “lectin” pathway (see FIG. 1 ). Complementtypically is activated or triggered by 1 of these 3 pathways, which asshown in FIG. 1 , converge at C3 activation. In a fourthcomplement-activation mechanism, referred to as the intrinsic pathway,serine proteases associated with the coagulation/fibrinolytic cascadeactivate the complement system directly through cleavage of C3 or C5,independently of the classical, alternate, and lectin pathways. Thesepathways can be distinguished by the process that initiates complementactivation. The classical pathway is initiated by antibody-antigencomplexes or aggregated forms of immunoglobulins; the alternativepathway is initiated by the recognition of structures on microbial andcell surfaces; and the lectin pathway, which is an antibody-independentpathway, is initiated by the binding of mannan binding lectin (MBL, alsodesignated mannose binding protein) to carbohydrates such as those thatare displayed on the surface of bacteria or viruses. Activation of thecascades results in production of complexes involved in proteolysis orcell lysis and peptides involved in opsonization, anaphylaxis andchemotaxis.

The complement cascade, which is a central component of an animal'simmune response, is an irreversible cascade. Numerous protein cofactorsregulate the process. Inappropriate regulation, typically inappropriateactivation, of the process can be a facet of or can occur in a varietyof disorders that involve inappropriate inflammatory and immuneresponses, such as those observed in acute and chronic inflammatorydiseases and other conditions involving an inappropriate immuneresponse. These diseases and disorders include autoimmune diseases, suchas rheumatoid arthritis and lupus, cardiac disorders and otherinflammatory diseases, such as sepsis and ischemia-reperfusion injury.

Because of the involvement of the complement pathways in a variety ofdiseases and conditions, components of the complement pathways aretargets for therapeutic intervention, particularly for inhibition of thepathway. Examples of such therapeutics include synthetic and naturalsmall molecule therapeutics, antibody inhibitors, and recombinantsoluble forms of membrane complement regulators. There are limitationsto strategies for preparing such therapeutics. Small molecules haveshort half-lives in vivo and need to be continually infused to maintaincomplement inhibition thereby limiting their role, especially in chronicdiseases. Therapeutic antibodies can result in an immune response in asubject, and thus can lead to complications in treatment, particularlytreatments designed to modulate immune responses. Thus, there exists aneed for therapeutics for treatment of complement-mediated diseases anddiseases in which complement activation plays a role. These includeacute and chronic inflammatory diseases. Accordingly, among theobjectives herein, it is an objective to provide such therapeutics totarget the activation of the complement cascade and to providetherapeutics and methods of treatment of diseases.

SUMMARY

Modified MTSP-1 polypeptides, comprising one or more of amino acidmodifications I41S, Q38H, D60bT, F60eS or R, Y60gW, ins97aV, D96K, F97G,G151H or N and Q192T, whereby the modified MTSP-1 polypeptide hasincreased activity and/or specificity for a complement protein comparedto the unmodified active form of the MTSP-1 polypeptide, where the aminoacid modifications are selected from among replacements, insertions, anddeletions in the primary amino acid sequence of the unmodified MTSP-1polypeptide; the modified MTSP-1 polypeptide cleaves a complementprotein to thereby inhibit or reduce complement activation compared toan active form of the unmodified MTSP-1 polypeptide that does notcontain the amino acid modification(s); residues are numbered bychymotrypsin numbering; corresponding residues are determined byalignment and chymotrypsin numbering; the unmodified MTSP-1 polypeptidecomprises the sequence of amino acids set forth in any of SEQ IDNOs.:1-4 (wild-type full-length MTSP-1, wild-type protease domainMTSP-1, wild-type mature MTSP-1, full-length MTSP-1 with C122S, proteasedomain MTSP-1 with C122S, mature MTSP-1 with C122S) or a catalyticallyactive fragment or form thereof that includes the amino acidmodification(s). Modifications are in the primary sequence, and includeinsertions, replacements and deletions. The modified MTSP-1 polypeptidesinclude modifications that improve or alter activity, and otherproperties, including properties that improve their use aspharmaceuticals. For example, the modified MTSP-1 polypeptides can beconjugated to moieties that increase stability, serum half-life,shelf-life and other such properties. These modifications includeconjugation to polymers, such as PEGylation moieties, and otherpolypeptides for targeting, identifying and purifying, to the modifiedMTPS-1.

The complement protein for which modified MTSP-1 polypeptides providedherein are modified to inactivate is C3, such that the modified MTSP-1polypeptides cleave a site that inactivates C3. By virtue ofinactivation of C3, complement activation is reduced or inhibited. Byvirtue of inhibition or reduction of complement activation, any disease,condition or disorder in which complement plays a role or in which areduction of complement activation can treat or reduce symptoms orpathology of the disease or disorder, can be treated with the modifiedMTSP-1 polypeptides provided herein. Target sites in C3 for inactivationcleavage include residues 737-744; cleavage within these residues, suchas between residues 740 and 741 of SEQ ID NO:9 (Q H A R↓A S H L),inactivates C3. Activity/specificity for the cleavage of C3 can beincreased compared to an active form of the unmodified MTSP-1polypeptide that does not contain the amino acid modification(s). Themodified MTSP-1 polypeptides provided herein are designed to haveincreased activity for cleavage of C3 that is least 1-fold greater ormore than 1-fold greater than an active form, such as the full length orprotease domain, of the unmodified MTSP-1 polypeptide of SEQ ID NO:4(the protease domain with the free cysteine, C122 by chymotrypsinnumbering, replaced by S). Cleavage activity for inactivating C3 can beincreased by any amount, such as at least 0.5-fold, 1-fold, 1.2 fold,1.5-fold, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or more than theunmodified modified MTSP-1 polypeptide of SEQ ID NO: 4. The unmodifiedMTSP-1 polypeptide is selected from among polypeptides of SEQ ID NOs.:1-4 and catalytically active portions thereof.

The modified MTSP-1 polypeptide, which includes at least onemodification, such I41S, Q38H, D60bT, F60eS or R, Y60gW, ins97aV, D96K,F97G, G151H or N and Q192T, or combinations of these or otherreplacements as described herein, has at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with the polypeptides of any of SEQ ID NOs.: 1-4. The modifiedMTSP-1 polypeptide can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 modifications,including insertions, deletions, and replacements in the primarysequence in the polypeptides of any of SEQ ID NOs:1-4 and catalyticallyactive portions thereof.

For example, provided are modified MTSP-1 polypeptides that comprise amodification corresponding to any one or more of I41S, Q38H, D60bT,F60eS, Y60gW, ins97aV, D96K, F97G, G151H, G151N, Q192T, Q192D and/orQ192E. The modified MTSP-1 polypeptides further can include additionalreplacements corresponding to and selected from one or more of F60eR,Y59F, F99L, T98P, Q175L or selected from one or more modifications at aposition corresponding to D217, such as D217V, I, L, W or M, and I41D,E, T, G or R. As above, corresponding positions are determined bychymotrypsin numbering. Exemplary modified MTSP-1 polypeptides includemodified MTSP-1 polypeptides that contain I41E/F99L/C122S/G151N/Q192T;I41D/C122S/G151N/Q192T; I41S/F99L/C122S/G151N/Q192V; orI41E/F99L/C122S/G151N/Q192T; or the same modifications except C122S isC122C. Other exemplary modified MTSP-1 polypeptide include modificationscorresponding to any of:

-   -   I41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192E, or    -   I41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192D, or    -   I41D/Y59F/D96E/F99L/C122S/G151N/Q192T, or    -   I41D/Y59F/C122S/G151N/Q192T.

Also provided are modified MTSP-1 polypeptides that includemodifications corresponding to any of:

-   -   I41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192E, or    -   Q38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99L/C122S/G151N/Q175        L/Q192D, or    -   Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/C122S/G151N/Q175        L/Q192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192E,        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/C122S/G151H/Q175        L/Q192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C122S/G151N/Q175L/Q192D,        or    -   Q38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99L/C122S/G151H/Q175        L/Q192D, or    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97Y/ins97aN/T98G/F99L/C122S/G151N/Q175L/Q        192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F99L/C122S/G151H/Q175L/Q        192D, or    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96P/F97W/ins97aN/T98G/F99L/C122S/G151N/Q175L/Q192E,        or    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C122S/G151N/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97E/ins97aV/T98G/F99L/C122S/G151H/Q175L/Q        192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96L/F97D/ins97aG/T98N/F99L/C122S/G151H/Q175L/Q        192E, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q        192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q        192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F99L/C122S/G151N/Q175L/Q        192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L,        or    -   I41E/F99L/C122S/G151N/Q192T, or 141D/C122S/G151N/Q192T, or    -   I41S/F99L/C122S/G151N/Q192V, or 141E/F99L/C122S/G151N/Q192T, or    -   I41D/Y59F/D96E/F99L/C122S/G151N/Q192T, or    -   I41D/Y59F/C122S/G151N/Q192T, or    -   I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175        L/Q192D, or    -   Q38H/I41S/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/F60eS/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/T98P/F99L/C122S/G151H/Q175L/Q        192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/F99L/C122S/G151H/Q175        L/Q192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/C122S/G151H/Q175        L/Q192D, or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192D,        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q192D,        or    -   Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192D, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D, or    -   Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192D, or    -   Q38H/I41S/D60bY/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D/D217V,        or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192G/D217V, or    -   I41S/D60bY/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D/D217V, or    -   I41S/D96M/F97G/ins97aV/T98P/F99L/C122S/Q192G/D217V, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192V/D217I, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192H, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192N/D217V, or    -   I41S/D60bY/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192D, or    -   Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192G/D217V, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192V, or    -   I41S/P49S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192G/D217V, or    -   I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192N/D217V, or    -   I41T/F97W/F99L/C122S/G151N/Q175M/Q192G/D217L, or    -   I41G/F97L/F99L/C122S/Q175A/Q192T/D217V, or    -   I41G/F97V/F99L/C122S/G151Q/Q175M/Q192A/D217L, or    -   I41G/F97I/F99L/C122S/G151L/Q175M/Q192S/D217V, or    -   I41G/F97S/F99L/C122S/G151N/Q175L/Q192G/D217I, or    -   I41T/F97L/F99L/C122S/G151N/Q175S/Q192S/D217W, or    -   I41D/F97T/F99M/C122S/Q192V/D217M, or the same modifications        except C122S is not modified and is C122C.

Also provided are modified MTSP-1 polypeptides that includemodifications selected from among combinations that include one or moreof modifications Q38H, I41S, D60bT, F60eS, Y60gW, D96K, F97G, Ins97aVand G151H as follows: G151H, Ins97aV, Ins97aV/G151H, F97G, F97G/G151H,F97G/Ins97aV, F97G/Ins97aV/G151H, D96K, D96K/G151H, D96K/Ins97aV,D96K/Ins97aV/G151H, D96K/F97G, D96K/F97G/G151H, D96K/F97G/Ins97aV,D96K/F97G/Ins97aV/G151H, Y60gW, Y60gW/G151H, Y60gW/Ins97aV,Y60gW/Ins97aV/G151H, Y60gW/F97G, Y60gW/F97G/G151H, Y60gW/F97G/Ins97aV,Y60gW/F97G/Ins97aV/G151H, Y60gW/D96K, Y60gW/D96K/G151H,Y60gW/D96K/Ins97aV, Y60gW/D96K/Ins97aV/G151H, Y60gW/D96K/F97G,Y60gW/D96K/F97G/G151H, Y60gW/D96K/F97G/Ins97aV,Y60gW/D96K/F97G/Ins97aV/G151H, F60eS, F60eS/G151H, F60eS/Ins97aV,F60eS/Ins97aV/G151H, F60eS/F97G, F60eS/F97G/G151H, F60eS/F97G/Ins97aV,F60eS/F97G/Ins97aV/G151H, F60eS/D96K, F60eS/D96K/G151H,F60eS/D96K/Ins97aV, F60eS/D96K/Ins97aV/G151H, F60eS/D96K/F97G,F60eS/D96K/F97G/G151H, F60eS/D96K/F97G/Ins97aV,F60eS/D96K/F97G/Ins97aV/G151H, F60eS/Y60gW, F60eS/Y60gW/G151H,F60eS/Y60gW/Ins97aV, F60eS/Y60gW/Ins97aV/G151H, F60eS/Y60gW/F97G,F60eS/Y60gW/F97G/G151H, F60eS/Y60gW/F97G/Ins97aV,F60eS/Y60gW/F97G/Ins97aV/G151H, F60eS/Y60gW/D96K,F60eS/Y60gW/D96K/G151H, F60eS/Y60gW/D96K/Ins97aV,F60eS/Y60gW/D96K/Ins97aV/G151H, F60eS/Y60gW/D96K/F97G,F60eS/Y60gW/D96K/F97G/G151H, F60eS/Y60gW/D96K/F97G/Ins97aV,F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, D60bT, D60bT/G151H, D60bT/Ins97aV,D60bT/Ins97aV/G151H, D60bT/F97G, D60bT/F97G/G151H, D60bT/F97G/Ins97aV,D60bT/F97G/Ins97aV/G151H, D60bT/D96K, D60bT/D96K/G151H,D60bT/D96K/Ins97aV, D60bT/D96K/Ins97aV/G151H, D60bT/D96K/F97G,D60bT/D96K/F97G/G151H, D60bT/D96K/F97G/Ins97aV,D60bT/D96K/F97G/Ins97aV/G151H, D60bT/Y60gW, D60bT/Y60gW/G151H,D60bT/Y60gW/Ins97aV, D60bT/Y60gW/Ins97aV/G151H, D60bT/Y60gW/F97G,D60bT/Y60gW/F97G/G151H, D60bT/Y60gW/F97G/Ins97aV,D60bT/Y60gW/F97G/Ins97aV/G151H, D60bT/Y60gW/D96K,D60bT/Y60gW/D96K/G151H, D60bT/Y60gW/D96K/Ins97aV,D60bT/Y60gW/D96K/Ins97aV/G151H, D60bT/Y60gW/D96K/F97G,D60bT/Y60gW/D96K/F97G/G151H, D60bT/Y60gW/D96K/F97G/Ins97aV,D60bT/Y60gW/D96K/F97G/Ins97aV/G151H, D60bT/F60eS, D60bT/F60eS/G151H,D60bT/F60eS/Ins97aV, D60bT/F60eS/Ins97aV/G151H, D60bT/F60eS/F97G,D60bT/F60eS/F97G/G151H, D60bT/F60eS/F97G/Ins97aV,D60bT/F60eS/F97G/Ins97aV/G151H, D60bT/F60eS/D96K,D60bT/F60eS/D96K/G151H, D60bT/F60eS/D96K/Ins97aV,D60bT/F60eS/D96K/Ins97aV/G151H, D60bT/F60eS/D96K/F97G,D60bT/F60eS/D96K/F97G/G151H, D60bT/F60eS/D96K/F97G/Ins97aV,D60bT/F60eS/D96K/F97G/Ins97aV/G151H, D60bT/F60eS/Y60gW,D60bT/F60eS/Y60gW/G151H, D60bT/F60eS/Y60gW/Ins97aV,D60bT/F60eS/Y60gW/Ins97aV/G151H, D60bT/F60eS/Y60gW/F97G,D60bT/F60eS/Y60gW/F97G/G151H, D60bT/F60eS/Y60gW/F97G/Ins97aV,D60bT/F60eS/Y60gW/F97G/Ins97aV/G151H, D60bT/F60eS/Y60gW/D96K,D60bT/F60eS/Y60gW/D96K/G151H, D60bT/F60eS/Y60gW/D96K/Ins97aV,D60bT/F60eS/Y60gW/D96K/Ins97aV/G151H, D60bT/F60eS/Y60gW/D96K/F97G,D60bT/F60eS/Y60gW/D96K/F97G/G151H, D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV,D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, I41S, I41S/G151H,I41S/Ins97aV, I41S/Ins97aV/G151H, I41S/F97G, I41S/F97G/G151H,I41S/F97G/Ins97aV, I41S/F97G/Ins97aV/G151H, I41S/D96K, I41S/D96K/G151H,I41S/D96K/Ins97aV, I41S/D96K/Ins97aV/G151H, I41S/D96K/F97G,I41S/D96K/F97G/G151H, I41S/D96K/F97G/Ins97aV,I41S/D96K/F97G/Ins97aV/G151H, 141S/Y60gW, 141S/Y60gW/G151H,I41S/Y60gW/Ins97aV, 141S/Y60gW/Ins97aV/G151H, 141S/Y60gW/F97G,I41S/Y60gW/F97G/G151H, 141S/Y60gW/F97G/Ins97aV,I41S/Y60gW/F97G/Ins97aV/G151H, 141S/Y60gW/D96K, I41S/Y60gW/D96K/G151H,141S/Y60gW/D96K/Ins97aV, I41S/Y60gW/D96K/Ins97aV/G151H,141S/Y60gW/D96K/F97G, I41S/Y60gW/D96K/F97G/G151H,141S/Y60gW/D96K/F97G/Ins97aV, I41S/Y60gW/D96K/F97G/Ins97aV/G151H,141S/F60eS, 141S/F60eS/G151H, I41S/F60eS/Ins97aV,141S/F60eS/Ins97aV/G151H, 141S/F60eS/F97G, I41S/F60eS/F97G/G151H,141S/F60eS/F97G/Ins97aV, I41S/F60eS/F97G/Ins97aV/G151H, 141S/F60eS/D96K,141S/F60eS/D96K/G151H, I41S/F60eS/D96K/Ins97aV,141S/F60eS/D96K/Ins97aV/G151H, I41S/F60eS/D96K/F97G,141S/F60eS/D96K/F97G/G151H, I41S/F60eS/D96K/F97G/Ins97aV,141S/F60eS/D96K/F97G/Ins97aV/G151H, I41S/F60eS/Y60gW,141S/F60eS/Y60gW/G151H, 141S/F60eS/Y60gW/Ins97aV,I41S/F60eS/Y60gW/Ins97aV/G151H, 141S/F60eS/Y60gW/F97G,I41S/F60eS/Y60gW/F97G/G151H, 141S/F60eS/Y60gW/F97G/Ins97aV,I41S/F60eS/Y60gW/F97G/Ins97aV/G151H, 141S/F60eS/Y60gW/D96K,I41S/F60eS/Y60gW/D96K/G151H, 141S/F60eS/Y60gW/D96K/Ins97aV,I41S/F60eS/Y60gW/D96K/Ins97aV/G151H, 141S/F60eS/Y60gW/D96K/F97G,I41S/F60eS/Y60gW/D96K/F97G/G151H, 141S/F60eS/Y60gW/D96K/F97G/Ins97aV,I41S/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, 141S/D60bT, I41S/D60bT/G151H,141S/D60bT/Ins97aV, 141S/D60bT/Ins97aV/G151H, I41S/D60bT/F97G,141S/D60bT/F97G/G151H, 141S/D60bT/F97G/Ins97aV,I41S/D60bT/F97G/Ins97aV/G151H, 141S/D60bT/D96K, 141S/D60bT/D96K/G151H,I41S/D60bT/D96K/Ins97aV, 141S/D60bT/D96K/Ins97aV/G151H,I41S/D60bT/D96K/F97G, 141S/D60bT/D96K/F97G/G151H,I41S/D60bT/D96K/F97G/Ins97aV, 141S/D60bT/D96K/F97G/Ins97aV/G151H,I41S/D60bT/Y60gW, 141S/D60bT/Y60gW/G151H, 141S/D60bT/Y60gW/Ins97aV,I41S/D60bT/Y60gW/Ins97aV/G151H, 141S/D60bT/Y60gW/F97G,I41S/D60bT/Y60gW/F97G/G151H, 141S/D60bT/Y60gW/F97G/Ins97aV,I41S/D60bT/Y60gW/F97G/Ins97aV/G151H, 141S/D60bT/Y60gW/D96K,I41S/D60bT/Y60gW/D96K/G151H, 141S/D60bT/Y60gW/D96K/Ins97aV,I41S/D60bT/Y60gW/D96K/Ins97aV/G151H, 141S/D60bT/Y60gW/D96K/F97G,I41S/D60bT/Y60gW/D96K/F97G/G151H, I41S/D60bT/Y60gW/D96K/F97G/Ins97aV,I41S/D60bT/Y60gW/D96K/F97G/Ins97aV/G151H, 141S/D60bT/F60eS,I41S/D60bT/F60eS/G151H, 141S/D60bT/F60eS/Ins97aV,I41S/D60bT/F60eS/Ins97aV/G151H, 141S/D60bT/F60eS/F97G,I41S/D60bT/F60eS/F97G/G151H, 141S/D60bT/F60eS/F97G/Ins97aV,I41S/D60bT/F60eS/F97G/Ins97aV/G151H, 141S/D60bT/F60eS/D96K,I41S/D60bT/F60eS/D96K/G151H, 141S/D60bT/F60eS/D96K/Ins97aV,I41S/D60bT/F60eS/D96K/Ins97aV/G151H, 141S/D60bT/F60eS/D96K/F97G,I41S/D60bT/F60eS/D96K/F97G/G151H, 141S/D60bT/F60eS/D96K/F97G/Ins97aV,I41S/D60bT/F60eS/D96K/F97G/Ins97aV/G151H, 141S/D60bT/F60eS/Y60gW,I41S/D60bT/F60eS/Y60gW/G151H, 141S/D60bT/F60eS/Y60gW/Ins97aV,I41S/D60bT/F60eS/Y60gW/Ins97aV/G151H, 141S/D60bT/F60eS/Y60gW/F97G,I41S/D60bT/F60eS/Y60gW/F97G/G151H, I41S/D60bT/F60eS/Y60gW/F97G/Ins97aV,I41S/D60bT/F60eS/Y60gW/F97G/Ins97aV/G151H, I41S/D60bT/F60eS/Y60gW/D96K,141S/D60bT/F60eS/Y60gW/D96K/G151H, I41S/D60bT/F60eS/Y60gW/D96K/Ins97aV,I41S/D60bT/F60eS/Y60gW/D96K/Ins97aV/G151H,I41S/D60bT/F60eS/Y60gW/D96K/F97G,I41S/D60bT/F60eS/Y60gW/D96K/F97G/G151H,I41S/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV,I41S/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H, Q38H/G151H,Q38H/Ins97aV, Q38H/Ins97aV/G151H, Q38H/F97G, Q38H/F97G/G151H,Q38H/F97G/Ins97aV, Q38H/F97G/Ins97aV/G151H, Q38H/D96K, Q38H/D96K/G151H,Q38H/D96K/Ins97aV, Q38H/D96K/Ins97aV/G151H, Q38H/D96K/F97G,Q38H/D96K/F97G/G151H, Q38H/D96K/F97G/Ins97aV,Q38H/D96K/F97G/Ins97aV/G151H, Q38H/Y60gW, Q38H/Y60gW/G151H,Q38H/Y60gW/Ins97aV, Q38H/Y60gW/Ins97aV/G151H, Q38H/Y60gW/F97G,Q38H/Y60gW/F97G/G151H, Q38H/Y60gW/F97G/Ins97aV,Q38H/Y60gW/F97G/Ins97aV/G151H, Q38H/Y60gW/D96K, Q38H/Y60gW/D96K/G151H,Q38H/Y60gW/D96K/Ins97aV, Q38H/Y60gW/D96K/Ins97aV/G151H,Q38H/Y60gW/D96K/F97G, Q38H/Y60gW/D96K/F97G/G151H,Q38H/Y60gW/D96K/F97G/Ins97aV, Q38H/Y60gW/D96K/F97G/Ins97aV/G151H,Q38H/F60eS, Q38H/F60eS/G151H, Q38H/F60eS/Ins97aV,Q38H/F60eS/Ins97aV/G151H, Q38H/F60eS/F97G, Q38H/F60eS/F97G/G151H,Q38H/F60eS/F97G/Ins97aV, Q38H/F60eS/F97G/Ins97aV/G151H, Q38H/F60eS/D96K,Q38H/F60eS/D96K/G151H, Q38H/F60eS/D96K/Ins97aV,Q38H/F60eS/D96K/Ins97aV/G151H, Q38H/F60eS/D96K/F97G,Q38H/F60eS/D96K/F97G/G151H, Q38H/F60eS/D96K/F97G/Ins97aV,Q38H/F60eS/D96K/F97G/Ins97aV/G151H, Q38H/F60eS/Y60gW,Q38H/F60eS/Y60gW/G151H, Q38H/F60eS/Y60gW/Ins97aV,Q38H/F60eS/Y60gW/Ins97aV/G151H, Q38H/F60eS/Y60gW/F97G,Q38H/F60eS/Y60gW/F97G/G151H, Q38H/F60eS/Y60gW/F97G/Ins97aV,Q38H/F60eS/Y60gW/F97G/Ins97aV/G151H, Q38H/F60eS/Y60gW/D96K,Q38H/F60eS/Y60gW/D96K/G151H, Q38H/F60eS/Y60gW/D96K/Ins97aV,Q38H/F60eS/Y60gW/D96K/Ins97aV/G151H, Q38H/F60eS/Y60gW/D96K/F97G,Q38H/F60eS/Y60gW/D96K/F97G/G151H, Q38H/F60eS/Y60gW/D96K/F97G/Ins97aV,Q38H/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H/D60bT, Q38H/D60bT/G151H,Q38H/D60bT/Ins97aV, Q38H/D60bT/Ins97aV/G151H, Q38H/D60bT/F97G,Q38H/D60bT/F97G/G151H, Q38H/D60bT/F97G/Ins97aV,Q38H/D60bT/F97G/Ins97aV/G151H, Q38H/D60bT/D96K, Q38H/D60bT/D96K/G151H,Q38H/D60bT/D96K/Ins97aV, Q38H/D60bT/D96K/Ins97aV/G151H,Q38H/D60bT/D96K/F97G, Q38H/D60bT/D96K/F97G/G151H,Q38H/D60bT/D96K/F97G/Ins97aV, Q38H/D60bT/D96K/F97G/Ins97aV/G151H,Q38H/D60bT/Y60gW, Q38H/D60bT/Y60gW/G151H, Q38H/D60bT/Y60gW/Ins97aV,Q38H/D60bT/Y60gW/Ins97aV/G151H, Q38H/D60bT/Y60gW/F97G,Q38H/D60bT/Y60gW/F97G/G151H, Q38H/D60bT/Y60gW/F97G/Ins97aV,Q38H/D60bT/Y60gW/F97G/Ins97aV/G151H, Q38H/D60bT/Y60gW/D96K,Q38H/D60bT/Y60gW/D96K/G151H, Q38H/D60bT/Y60gW/D96K/Ins97aV,Q38H/D60bT/Y60gW/D96K/Ins97aV/G151H, Q38H/D60bT/Y60gW/D96K/F97G,Q38H/D60bT/Y60gW/D96K/F97G/G151H, Q38H/D60bT/Y60gW/D96K/F97G/Ins97aV,Q38H/D60bT/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H/D60bT/F60eS,Q38H/D60bT/F60eS/G151H, Q38H/D60bT/F60eS/Ins97aV,Q38H/D60bT/F60eS/Ins97aV/G151H, Q38H/D60bT/F60eS/F97G,Q38H/D60bT/F60eS/F97G/G151H, Q38H/D60bT/F60eS/F97G/Ins97aV,Q38H/D60bT/F60eS/F97G/Ins97aV/G151H, Q38H/D60bT/F60eS/D96K,Q38H/D60bT/F60eS/D96K/G151H, Q38H/D60bT/F60eS/D96K/Ins97aV,Q38H/D60bT/F60eS/D96K/Ins97aV/G151H, Q38H/D60bT/F60eS/D96K/F97G,Q38H/D60bT/F60eS/D96K/F97G/G151H, Q38H/D60bT/F60eS/D96K/F97G/Ins97aV,Q38H/D60bT/F60eS/D96K/F97G/Ins97aV/G151H, Q38H/D60bT/F60eS/Y60gW,Q38H/D60bT/F60eS/Y60gW/G151H, Q38H/D60bT/F60eS/Y60gW/Ins97aV,Q38H/D60bT/F60eS/Y60gW/Ins97aV/G151H, Q38H/D60bT/F60eS/Y60gW/F97G,Q38H/D60bT/F60eS/Y60gW/F97G/G151H, Q38H/D60bT/F60eS/Y60gW/F97G/Ins97aV,Q38H/D60bT/F60eS/Y60gW/F97G/Ins97aV/G151H, Q38H/D60bT/F60eS/Y60gW/D96K,Q38H/D60bT/F60eS/Y60gW/D96K/G151H, Q38H/D60bT/F60eS/Y60gW/D96K/Ins97aV,Q38H/D60bT/F60eS/Y60gW/D96K/Ins97aV/G151H,Q38H/D60bT/F60eS/Y60gW/D96K/F97G,Q38H/D60bT/F60eS/Y60gW/D96K/F97G/G151H,Q38H/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV,Q38H/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H/I41S,Q38H/I41S/G151H, Q38H/I41S/Ins97aV, Q38H/I41S/Ins97aV/G151H,Q38H/I41S/F97G, Q38H/I41S/F97G/G151H, Q38H/I41S/F97G/Ins97aV,Q38H/I41S/F97G/Ins97aV/G151H, Q38H/I41S/D96K, Q38H/I41S/D96K/G151H,Q38H/I41S/D96K/Ins97aV, Q38H/I41S/D96K/Ins97aV/G151H,Q38H/I41S/D96K/F97G, Q38H/I41S/D96K/F97G/G151H,Q38H/I41S/D96K/F97G/Ins97aV, Q38H/I41S/D96K/F97G/Ins97aV/G151H,Q38H/I41S/Y60gW, Q38H/I41S/Y60gW/G151H, Q38H/I41S/Y60gW/Ins97aV,Q38H/I41S/Y60gW/Ins97aV/G151H, Q38H/I41S/Y60gW/F97G,Q38H/I41S/Y60gW/F97G/G151H, Q38H/I41S/Y60gW/F97G/Ins97aV,Q38H/I41S/Y60gW/F97G/Ins97aV/G151H, Q38H/I41S/Y60gW/D96K,Q38H/I41S/Y60gW/D96K/G151H, Q38H/I41S/Y60gW/D96K/Ins97aV,Q38H/I41S/Y60gW/D96K/Ins97aV/G151H, Q38H/I41S/Y60gW/D96K/F97G,Q38H/I41S/Y60gW/D96K/F97G/G151H, Q38H/I41S/Y60gW/D96K/F97G/Ins97aV,Q38H/I41S/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H/I41S/F60eS,Q38H/I41S/F60eS/G151H, Q38H/I41S/F60eS/Ins97aV,Q38H/I41S/F60eS/Ins97aV/G151H, Q38H/I41S/F60eS/F97G,Q38H/I41S/F60eS/F97G/G151H, Q38H/I41S/F60eS/F97G/Ins97aV,Q38H/I41S/F60eS/F97G/Ins97aV/G151H, Q38H/I41S/F60eS/D96K,Q38H/I41S/F60eS/D96K/G151H, Q38H/I41S/F60eS/D96K/Ins97aV,Q38H/I41S/F60eS/D96K/Ins97aV/G151H, Q38H/I41S/F60eS/D96K/F97G,Q38H/I41S/F60eS/D96K/F97G/G151H, Q38H/I41S/F60eS/D96K/F97G/Ins97aV,Q38H/I41S/F60eS/D96K/F97G/Ins97aV/G151H, Q38H/I41S/F60eS/Y60gW,Q38H/I41S/F60eS/Y60gW/G151H, Q38H/I41S/F60eS/Y60gW/Ins97aV,Q38H/I41S/F60eS/Y60gW/Ins97aV/G151H, Q38H/I41S/F60eS/Y60gW/F97G,Q38H/I41S/F60eS/Y60gW/F97G/G151H, Q38H/I41S/F60eS/Y60gW/F97G/Ins97aV,Q38H/I41S/F60eS/Y60gW/F97G/Ins97aV/G151H, Q38H/I41S/F60eS/Y60gW/D96K,Q38H/I41S/F60eS/Y60gW/D96K/G151H, Q38H/I41S/F60eS/Y60gW/D96K/Ins97aV,Q38H/I41S/F60eS/Y60gW/D96K/Ins97aV/G151H,Q38H/I41S/F60eS/Y60gW/D96K/F97G, Q38H/I41S/F60eS/Y60gW/D96K/F97G/G151H,Q38H/I41S/F60eS/Y60gW/D96K/F97G/Ins97aV,Q38H/I41S/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H/I41S/D60bT,Q38H/I41S/D60bT/G151H, Q38H/I41S/D60bT/Ins97aV,Q38H/I41S/D60bT/Ins97aV/G151H, Q38H/I41S/D60bT/F97G,Q38H/I41S/D60bT/F97G/G151H, Q38H/I41S/D60bT/F97G/Ins97aV,Q38H/I41S/D60bT/F97G/Ins97aV/G151H, Q38H/I41S/D60bT/D96K,Q38H/I41S/D60bT/D96K/G151H, Q38H/I41S/D60bT/D96K/Ins97aV,Q38H/I41S/D60bT/D96K/Ins97aV/G151H, Q38H/I41S/D60bT/D96K/F97G,Q38H/I41S/D60bT/D96K/F97G/G151H, Q38H/I41S/D60bT/D96K/F97G/Ins97aV,Q38H/I41S/D60bT/D96K/F97G/Ins97aV/G151H, Q38H/I41S/D60bT/Y60gW,Q38H/I41S/D60bT/Y60gW/G151H, Q38H/I41S/D60bT/Y60gW/Ins97aV,Q38H/I41S/D60bT/Y60gW/Ins97aV/G151H, Q38H/I41S/D60bT/Y60gW/F97G,Q38H/I41S/D60bT/Y60gW/F97G/G151H, Q38H/I41S/D60bT/Y60gW/F97G/Ins97aV,Q38H/I41S/D60bT/Y60gW/F97G/Ins97aV/G151H, Q38H/I41S/D60bT/Y60gW/D96K,Q38H/I41S/D60bT/Y60gW/D96K/G151H, Q38H/I41S/D60bT/Y60gW/D96K/Ins97aV,Q38H/I41S/D60bT/Y60gW/D96K/Ins97aV/G151H,Q38H/I41S/D60bT/Y60gW/D96K/F97G, Q38H/I41S/D60bT/Y60gW/D96K/F97G/G151H,Q38H/I41S/D60bT/Y60gW/D96K/F97G/Ins97aV,Q38H/I41S/D60bT/Y60gW/D96K/F97G/Ins97aV/G151H, Q38H/I41S/D60bT/F60eS,Q38H/I41S/D60bT/F60eS/G151H, Q38H/I41S/D60bT/F60eS/Ins97aV,Q38H/I41S/D60bT/F60eS/Ins97aV/G151H, Q38H/I41S/D60bT/F60eS/F97G,Q38H/I41S/D60bT/F60eS/F97G/G151H, Q38H/I41S/D60bT/F60eS/F97G/Ins97aV,Q38H/I41S/D60bT/F60eS/F97G/Ins97aV/G151H, Q38H/I41S/D60bT/F60eS/D96K,Q38H/I41S/D60bT/F60eS/D96K/G151H, Q38H/I41S/D60bT/F60eS/D96K/Ins97aV,Q38H/I41S/D60bT/F60eS/D96K/Ins97aV/G151H,Q38H/I41S/D60bT/F60eS/D96K/F97G, Q38H/I41S/D60bT/F60eS/D96K/F97G/G151H,Q38H/I41S/D60bT/F60eS/D96K/F97G/Ins97aV,Q38H/I41S/D60bT/F60eS/D96K/F97G/Ins97aV/G151H,Q38H/I41S/D60bT/F60eS/Y60gW, Q38H/I41S/D60bT/F60eS/Y60gW/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/Ins97aV,Q38H/I41S/D60bT/F60eS/Y60gW/Ins97aV/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/F97G,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/Ins97aV,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/Ins97aV/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/D96K,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/Ins97aV,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/Ins97aV/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/G151H,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV/G151H, and the samemodifications except C122S is not modified and is C122C.

Also provided are modified MTSP-1 polypeptides that includemodifications corresponding to any of: T98P/F99LQ175L/Q192D, optionallyC122S, and one or more selected from among: Q38H, I41S, D60bT, F60eS,Y60gW, D96K, F97G, Ins97aV and G151H as follows:T98P/F99L/C122S/G151H/Q175L/Q192D, 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Q38H/I41S/D60bT/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/D96K/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/D96K/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/D96K/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/D96K/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/D96K/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/D96K/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/D96K/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/D96K/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/Y60gW/D96K/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D, Q38H/I41S/D60bT/F60eS/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D ,Q38H/I41S/D60bT/F60eS/D96K/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/D96K/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D, Q38H/I41S/D60bT/F60eS/Y60gW/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/T98P/F99L/C122S/G151H/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV/T98P/F99L/C122S/Q175L/Q192D,Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/Ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,and the same modifications except C122S is not modified and is C122C.

Other exemplary modified MTSP-1 polypeptides can contain replacements,insertions, and/or deletions corresponding to:

-   -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/G151H/Q175L/Q192D,        including those in which the unmodified MTSP-1 polypeptide        comprises or is the protease domain of SEQ ID NO:2 or SEQ ID        NO:4. Other such exemplary modified MTSP-1 polypeptides include,        but are not limited to:    -   modified MTSP-1 polypeptides comprising modifications selected        from among combinations of modifications in which the protease        domain cleaves human C3 in vitro with an EC₅₀ of less than 10        nM:    -   ins97aA/F97G/T98L/C122S/Q175M/Q192A/D217I/K224R,    -   Q38Y/I41S/D60bT/F60eR/Y60gW/D96M/F97N/T98G/F99L/C122S/G151N/Q175L,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G151H/Q175L/Q192E,    -   Q38G/H40R/I41H/D60bN/F97D/F99L/C122S/Q175L/Q192G,    -   Q38Y/I41S/D60bT/F60eR/Y60gW/D96M/F97N/T98G/F99L/C122S/G151N/Q175L,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G151H/Q175L/Q192E,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96F/F97D/ins97aE/T98S/F99L/C122S/G151H/Q175L/Q192A,    -   Q38F/I41A/D60bT/F60eG/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E,    -   Q38H/I41S/D60bT/F60eS/Y60gW/ins97aV/F97D/T98P/F99L/C122S/G151H/Q175L,    -   Q38H/I41A/D60bV/F60eT/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aA/T98P/F99L/C122S/G151H/Q175L/Q192D,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G151N/Q175L/Q192E,    -   Q38Y/I41A/D60bL/F60eQ/ins97aV/F97D/T98P/F99L/C122S/G151N/Q175M/Q192A,    -   Q38H/I41S/D60bF/F60        eV/F97D/ins97aV/T98P/F99L/C122S/G151N/Q175L/Q192A,    -   Q38H/I41A/D60bV/F60eA/Y60gW/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192V,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96I/ins97aN/F97Y/T98G/F99L/C122S/G151H/Q175L/Q192D,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96L/ins97aG/F97D/T98N/F99L/C122S/G151H/Q175L/Q192E,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G151H/Q175L/Q192D,    -   Q38F/I41S/D60bF/F60eR/Y60gF/F97T/ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192V,    -   Q38H/I41S/D60bY/D96Y/ins97aV/F97D/T98P/F99L/L106M/C122S/I136M/Q192G/Q209L/D217T,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96S/ins97aR/F97A/T98S/F99L/C122S/G151N/Q175L/Q192T,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96I/ins97aN/F97Y/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   Q38H/I41A/D60bT/F60eH/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192D,    -   Q38H/I41A/D60bV/F60eR/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192D,    -   ins97aY/F97G/T98V/C122S/Q175M/Q192S/D217V,    -   Q38H/I41S/D60bS/ins97aV/F97D/T98P/F99L/M117L/C122S/I136T/Q192G/D217,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C122S/G151H/Q175L/Q192D,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192A,    -   H40R/I41H/F97D/F99L/C122S/Q175M/Q192G/D217V/K224Y,    -   I41G/F97I/F99L/C122S/G151L/Q175M/Q192S/D217V,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/T150S/G151H/Q175L/Q192D/Q209L,    -   Q38H/I41A/D60bV/F60eI/Y60gW/F97T/ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96I/F97N/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   Q38H/I41A/D60bW/ins97aV/F97D/T98P/F99L/C122S/I136M/Q192G/D217N,    -   Q38H/I41S/D60bF/F60eT/ins97aV/F97D/T98P/F99L/C122S/H143Q/G151N/Q175L/Q192G,    -   Q38Y/I41S/D60bT/Y60gW/D96M/F97N/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   Q38H/I41A/F60eH/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192A,    -   I41T/D60bW/F60eH/F97D/ins97aV/T98P/F99L/C122S/G151N/Q175L/Q192G,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96F/F97Y/ins97aD/T98G/F99L/C122S/G151H/Q175L/Q192D,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96F/F97S/ins97aH/T98G/F99L/C122S/G151N/Q175L/Q192G,    -   Q38H/I41S/D60bT/F60eS/Y60gW/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192E,    -   I41S/F99L/C122S/G151N/Q175M/Q192G/D217V,    -   Q38R/I41S/D60bY/F60eD/ins97aV/F97D/T98P/F99L/C122S/G151N/Q175M/Q192A,    -   Q38Y/I41S/D60bT/F60eR/Y60eR/Y60gW/D96M/T98G/F99L/C122S/G151N/Q175L/Q192D,    -   H40R/I41H/Y60gH/F97D/F99L/C122S/Q175M/Q192G/D217I/K224L,    -   Q38H/I41A/D60bV/F60eR/Y60gW/F97D/F99L/C122S/G151H/Q175L/Q192D,    -   Q38Y/I41S/D60bT/F60eR/Y60gW/D96M/F97N/T98G/F99L/C122S/Q175L/Q192D,    -   Q38H/I41A/D60bV/F60eR/Y60gW/D96F/F97Y/ins97an/T98G/F99M/C122S/G151N/Q175L/Q192G,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97N/ins97aE/T98S/F99L/C122S/G151H/Q175L/Q192D,    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F99L/C122S/G151N/Q175L/Q192D,        and the same modifications except C122S is not modified and is        C122C.

Exemplary of modified MTSP-1 polypeptides provided herein are those thatcontain or have the sequences set forth in any of SEQ ID NOs.: 6, 8,21-59 and 63-81. This includes the modified MTSP-1 polypeptide whosesequence is set forth in SEQ ID NO: 35 or in SEQ ID NO: 42. MTSP-1polypeptides that contain the modificationsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D(with or without the C122S modification) are exemplary of suchpolypeptides.

For all of the modified MTSP-1 polypeptides provided herein, theunmodified MTSP-1 polypeptides include those that contain or have thesequence of amino acid residues set forth in SEQ ID NO:2 or 4 (proteasedomain). Included are full-length, two chain forms, two chain activatedforms, and single chain forms that contain the protease domain or acatalytically active portion thereof.

The modified MTSP-1 polypeptides are selected to cleave and inactivateC3 so that, in vivo, complement activation is reduced. Modified MTSP-1polypeptides provided herein include those that cleave within residuesQHARASHL (residues 737-744) of human C3 (SEQ ID NO:9), such as those inwhich P1-P1′ is RA.

All or any of the modified MTSP-1 polypeptides provided herein caninclude structural modifications and post-translational modificationssuch as addition of a polymer(s) to increase serum half-life and/or toreduce immunogenicity or both. Structural modifications includealterations in glycosylation, such as addition of glycosylation sites ortypes of glycosylation, and additions of polymers, such as dextran,sialylation and PEGylation. Any of the modified MTSP-1 polypeptides canbe PEGylated to increase half-life and/or serum stability, particularlyfor indications in which extended duration of action is desired. Themodified MTSP-1 polypeptide can be further modified, such as by additionor elimination of lysines or other residues to alter PEGylation or byaddition of or elimination of glycosylation sites.

Also, provided are fusion proteins, containing a modified MTSP-1polypeptide or a catalytically active portion of a modified MTSP-1polypeptide provided herein fused or otherwise conjugated via a chemicalor physical linker or bond to a non-MTSP-1 protease polypeptide or aportion thereof. Exemplary of such non-protease polypeptides is amultimerization domain, such as an Fc domain, or a protein transductiondomain (PTD).

Also provided are nucleic acid molecules encoding any of the modifiedMTSP-1 polypeptides and fusion proteins provided herein. Vectorscontaining the nucleic acid molecules also are provided. The vectors canbe prokaryotic vectors or eukaryotic vectors, and include expressionvectors, viral vectors, and vectors for gene therapy. Viral vectorsinclude, but are not limited to, a herpes virus simplex vector, or avaccinia virus vector, or an adenoviral vector, or a retroviral vector,or an insect vector.

Provided are isolated cells, isolated non-human cells (that cannotdevelop into a zygote or into a human by any method available to thoseof skill in the art), and cell cultures that contain the nucleic acidmolecule or nucleic acid molecules or vectors encoding the modifiedMTSP-1 polypeptides provided herein. Methods for making the modifiedMTSP-1 polypeptides are provided, and include amino acid synthesismethods, and recombinant DNA methods. These include introducing anucleic acid or vector encoding a modified MTSP-1 polypeptide providedherein into a cell; culturing the cell under conditions whereby thepolypeptide is expressed; and, optionally, isolating or purifying theexpressed modified MTSP-1 polypeptide. Cells include any suitable cellsor cell lines, including eukaryotic cells or prokaryotic cells. Theseinclude bacterial cells, mammalian cells, and yeast cells. For example,the cell can be a CHO cell, a BHK cell, Saccharomyces, Pichia, and suchmammalian cells. Cells and methods for producing recombinant therapeuticpolypeptides are well known to those of skill in the art.

The nucleic acids, vectors, and polypeptides can be used for treating orin methods of treatment of a disease or condition mediated by orinvolving complement activation, wherein inhibition of complementactivation effects treatment or amelioration of the disease orcondition. The nucleic acids and vectors can be used for gene therapy,or the polypeptides can be administered. Suitable routes ofadministration include parenteral, local, systemic and transdermalroutes. These include intravenous, intramuscular, subcutaneous, andintravitreal administration.

Provided are methods of treating a disease or condition mediated by orinvolving complement activation where inhibition or reduction ofcomplement activation effects treatment or some amelioration of symptomsor prevents (reduces the risk) of such disease or condition. The nucleicacid or vector or polypeptide is administered to a subject to treat orprevent (reduce the risk or symptoms) the disease or condition.Complement-mediated diseases or disorders or conditions include, but arenot limited to, inflammatory disease and conditions, sepsis, rheumatoidarthritis (RA), a cardiovascular disease, membranoproliferativeglomerulonephritis (MPGN), ophthalmic or ocular diseases or disorders,multiple sclerosis (MS), myasthenia gravis (MG), asthma, inflammatorybowel disease, immune complex (IC)-mediated acute inflammatory tissueinjury, Alzheimer's Disease (AD), transplanted organ rejection, andischemia-reperfusion injury.

The disease or condition can be an ocular or ophthalmic disease orrejection or inflammation due to a transplanted organ. Exemplary of suchdiseases, disorders or conditions is a diabetic retinopathy or a maculardegeneration, including age-related macular degeneration (AMD) anddelayed renal graft function (DGF).

The polypeptides provided herein can be used for inhibiting complementactivation by contacting a modified MTSP-1 polypeptide with a complementprotein C3, whereby complement protein C3 is cleaved such thatcomplement activation is reduced or inhibited. The subjects fortreatment with the polypeptides, methods and uses herein can be anyanimal, including humans, and domesticated animals, particularly, dogs,cats and other pets and farm animals. Inhibition of complementactivation can reduce the risk of developing (prevent) a disease,condition or disorder or lessen the disease, condition or disorder if itdevelops, or treat the disease or disorder or condition. Inhibition ofcomplement activation, among its effects, leads to a reduction ofinflammatory symptoms associated with a complement-mediated disease ordisorder selected from among an inflammatory disorder, aneurodegenerative disorder, and a cardiovascular disorder. As notedabove, complement-mediated diseases or disorders or conditions include,but are not limited to, sepsis, Rheumatoid arthritis (RA), ocular orophthalmic disorders, membranoproliferative glomerulonephritis (MPGN),Multiple Sclerosis (MS), delayed rejection of or inflammation oftransplanted organs or tissues, Myasthenia gravis (MG), asthma,inflammatory bowel disease, immune complex (IC)-mediated acuteinflammatory tissue injury, Alzheimer's Disease (AD), andIschemia-reperfusion injury, and any others known to those of skill inthe art.

The complement-mediated disease, condition or disorder can result from atreatment of a subject. For example, ischemia-reperfusion injury can becaused by an event or treatment selected from among myocardial infarct(MI), stroke, angioplasty, coronary artery bypass graft, cardiopulmonarybypass (CPB), and hemodialysis. Treatment with a modified MTSP-1polypeptide provided herein can be effected prior to a treatment of asubject that results in or has risk of causing a complement mediateddisorder, condition or disease.

Provided are uses of the modified MTSP-1 polypeptides provided hereinand methods of treating a disease or condition mediated by or involvingcomplement activation, by administering a modified MTSP-1 polypeptideprovided herein, where inhibition of complement activation effectstreatment or amelioration of the disease or condition. The modifiedMTSP-1 polypeptides also can be administered to reduce the risk(prevent) of a developing a disease or condition or to reduce thesymptoms of such disease, disorder or condition development.Complement-mediated diseases, conditions, or disorders include any notedabove or below. Included are ocular or ophthalmic diseases or rejectionof or inflammation due to a transplanted organ. Such diseases andconditions include diabetic retinopathy and a macular degeneration, suchas AMD, and delayed renal graft function (DGF). The modified MTSP-1polypeptide (or encoding nucleic acid molecule or vector) can beadministered by any suitable route, including those discussed above andelsewhere herein, such as parenterally, such as administeredintravenously, or subcutaneously. For treatment of ophthalmic disordersthe modified MTSP-1 polypeptides can be administered locally, such as byintravitreal injection or by topical application to the eye. Themodified MTSP-1 polypeptide can be modified for introduction into theeye, such as by fusion to a protein transduction domain to facilitatetransduction into the vitreous humor. For any application, method, oruse, the modified MTSP-1 polypeptide can be modified to increase serumhalf-life, such as by PEGylation.

Also provided are combinations and kits, that include: (a) a modifiedMTSP-1 polypeptide provided herein; and (b) a second agent or agents fortreating a complement-mediated disease or disorder. Second agentsinclude, but are not limited to anti-inflammatory agent(s) andanticoagulant(s), such as, but not limited to, any one or more of anon-steroidal anti-inflammatory drug (NSAID), antimetabolite,corticosteroid, analgesic, cytotoxic agent, pro-inflammatory cytokineinhibitor, anti-inflammatory cytokines, B cell targeting agents,compounds targeting T antigens, adhesion molecule blockers, chemokinereceptor antagonists, kinase inhibitors, PPAR-γ (gamma) ligands,complement inhibitors, heparin, warfarin, acenocoumarol, phenindione,EDTA, citrate, oxalate, argatroban, lepirudin, bivalirudin, andximelagatran.

The methods, uses or combinations include those in which the modifiedMTSP-1 polypeptide comprises the modification I41D or I41S, particularlyI41S, and particularly modified MTSP-1 polypeptides containing themodificationsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D, orQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/G151H/Q175L/Q192Dor I41D/C122S/G151N/Q192T or I41D/G151N/Q192T. The replacement C122S(with reference to the protease domain) is included to eliminate a freecysteine to reduce aggregation. The unmodified MTSP-1 polypeptideincludes the protease domain, such as that of the sequence of amino acidresidues set forth in SEQ ID NO:4, such as a modified MTSP-1 polypeptidethat contains the sequence of amino acid residues set forth in any ofSEQ ID NOs:35 and 42.

Provided are methods of treating DGF by intravenously administering amodified MTSP-1 polypeptide provided herein, such as a modified MTSP-1polypeptide that contains the sequence of amino acid residues set forthin any of SEQ ID NOs:35 and 42 or a catalytically active portionthereof, such as a modified MTSP-1 polypeptide that comprises thereplacementsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D.Dosages include any suitable dose and any suitable dosage regimen. Asingle dosage includes 0.1 mg to 1 mg. Treatment can be once or can berepeated a plurality of times, such as every 2 days, 3 days, 4 days, 5days, 6 days, weekly, bi-weekly or monthly. The modified MTSP-1polypeptides can be modified, such as by PEGylation or multimerizationto effect increased half-life or increased bioavailability.

Provided are uses of the modified MTSP-1 polypeptides and/or methods oftreating an ophthalmic disorder or ocular disorder, by administering amodified MTSP-1 polypeptide provided herein such as systemically or tothe eye. Ophthalmic disorders include diabetic retinopathy and maculardegeneration, such as AMD. Suitable dosages can be empiricallydetermined, and include a single dosage that is 0.1 to 1 mg. Exemplaryof modified MTSP-1 polypeptides for use for these indications are anyprovided herein that cleave C3. These include modified MTSP-1polypeptides that contain the sequence of amino acid residues set forthin any of SEQ ID NOs:35 and 42 or a catalytically active portionthereof, such as, but not limited to, a modified MTSP-1 polypeptide thatcomprises the replacements, or insertions and/or deletions:

-   -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/G151H/Q175L/Q192D    -   or the modified MTSP-1 polypeptide comprises the replacements:        I41D/G151N/Q192T or 141D/C122S/G151N/Q192T. The polypeptides can        be administered to the eye as described above, such as by        intravitreal injection or other local method. Treatment can be        once or repeated a plurality of times, such as every 2 days, 3        days, 4 days, 5 days, 6 days, weekly, bi-weekly or monthly. The        modified MTSP-1 polypeptides include those of SEQ ID NO:35 or a        catalytically active portion thereof or full-length forms or        catalytically active portions thereof. The modified MTSP-1        polypeptides can be modified, such as by PEGylation or        multimerization to effect increased half-life or increased        bioavailability.

For any application, the modified MTSP-1 polypeptide can be a singlechain or two chain form of the modified MTSP-1 polypeptide or a zymogenform that is activated in vivo. The protease domains are active assingle chains. The modified MTSP-1 polypeptides can include othermodifications, such as PEGylation to alter or improve pharmacologicalproperties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an overview of the classical, lectin, and alternativecomplement pathways and the activation of the terminal complementcomplex, the membrane attack complex (MAC). The figure depicts many ofthe more than 30 proteins that participate in the complement cascade,their action within the cascade, and where applicable, their points ofconvergence among the complement pathways. For example, the threepathways converge upon the generation of a C3 convertase, which cleavesC3 to form a C5 convertase yielding the formation of the MAC. The figurealso depicts the generation of many of the complement cleavage products.

FIG. 2 depicts a table that provides the anti-C3 activity (as an ED50)of 14 variant MTSP-1 polypeptides, to assess the effects of eachmutation on the C3 cleavage activity of the modified MTSP-1 polypeptidecontaining the modificationsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D(see, SEQ ID NO:35), measured in vitro. Each of the modified MTSP-1polypeptides contain all of the modifications in the modified MTSP-1protease domain set forth in SEQ ID NO:35, except for the onemodification whose effect on anti-C3 activity is assessed. Cells in thetable with bold borders indicate that the amino acid residue at thatposition is the same residue as in the wild-type MTSP-1 protease domainset forth in SEQ ID NO:4. Cells without bold borders indicate that theamino acid residue at that position is the same residue as in themodified MTSP-1 protease domain set forth in SEQ ID NO:35.

DETAILED DESCRIPTION

Outline

-   -   A. DEFINITIONS    -   B. MTSP-1 STRUCTURE AND FUNCTION        -   1. Serine Proteases        -   2. Structure        -   3. Function/Activity    -   C. COMPLEMENT INHIBITION BY TARGETING C3        -   1. Complement Protein C3 and its Role in Initiating            Complement            -   a. Classical Pathway            -   b. Alternative Pathway            -   c. Lectin Pathway            -   d. Complement-Mediated Effector Functions                -   i. Complement-Mediated Lysis: Membrane Attack                    Complex                -   ii. Inflammation                -   iii. Chemotaxis                -   iv. Opsonization                -   v. Activation of the Humoral Immune Response        -   2. C3 Structure and Function            -   a. C3a            -   b. C3b                -   i. Inhibitors of C3b    -   D. MODIFIED MTSP-1 POLYPEPTIDES THAT CLEAVE C3        -   1. Exemplary Modified MTSP-1 Polypeptides        -   2. Additional Modifications            -   a. Decreased Immunogenicity            -   b. Fc Domains            -   c. Conjugation to Polymers            -   d. Protein Transduction Domains    -   E. ASSAYS TO ASSESS AND/OR MONITOR MTSP-1 ACTIVITY ON        COMPLEMENT-MEDIATED FUNCTIONS        -   1. Methods for Assessing MTSP-1 Activity and Specificity for            Cleaving Complement Protein C3 to Inactivate it            -   a. Protein Detection                -   i. SDS-PAGE Analysis                -   ii. Enzyme Immunoassay                -   iii. Radial Immunodiffusion (RID)            -   b. Hemolytic Assays            -   c. Methods for Determining Cleavage Sites        -   2. Methods for Assessing Wild Type MTSP-1 Activity            -   a. Cleavage of MTSP-1 Substrates            -   b. MTSP-1-Substrate Binding Assays            -   c. C3 Cleavage Assays        -   3. Specificity        -   4. Disease Models    -   F. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING MODIFIED MTSP-1        POLYPEPTIDES        -   1. Isolation or Preparation of Nucleic Acids Encoding MTSP-1            Polypeptides        -   2. Generation of Mutant or Modified Nucleic Acids and            Encoding Polypeptides        -   3. Vectors and Cells        -   4. Expression            -   a. Prokaryotic Cells            -   b. Yeast Cells            -   c. Insects and Insect Cells            -   d. Mammalian Expression            -   e. Plants        -   5. Purification        -   6. Additional Modifications            -   a. PEGylation            -   b. Fusion Proteins        -   7. Nucleic Acid Molecules    -   G. COMPOSITIONS, FORMULATIONS AND DOSAGES        -   1. Administration of Modified MTSP-1 Polypeptides        -   2. Administration of Nucleic Acids Encoding Modified MTSP-1            Polypeptides (Gene Therapy)    -   H. THERAPEUTIC USES AND METHODS OF TREATMENT        -   1. Disease Mediated by Complement Activation            -   a. Rheumatoid Arthritis            -   b. Sepsis            -   c. Multiple Sclerosis            -   d. Alzheimer's Disease            -   e. Ischemia-Reperfusion Injury            -   f. Ocular Disorders                -   Age-Related Macular Degeneration (AMD)            -   g. Organ Transplantation and Delayed Graft Function                (DGF)        -   2. Therapeutic Uses            -   a. Immune-Mediated Inflammatory Diseases            -   b. Neurodegenerative Disease            -   c. Cardiovascular Disease            -   d. Age-Related Macular Degeneration (AMD)            -   e. Organ Transplant                -   Delayed Graft Function        -   3. Combination Therapies    -   I. EXAMPLES

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GENBANK sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there is a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such identifier or address, it is understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information is known and can be readilyaccessed, such as by searching the internet and/or appropriatedatabases. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, cleavage refers to the breaking of peptide bonds by aprotease. The cleavage site motif for a protease involves residues N-and C-terminal to the scissile bond (the unprimed and primed sides,respectively, with the cleavage site for a protease defined as . . .P3-P2-P1-P1′-P2′-P3′ . . . , and cleavage occurs between the P1 and P1′residues). In human C3, cleavage by a C3 convertase occurs betweenresidues R and S (see residues 746-751 of SEQ ID NO: 9, cleavage betweenresidue 748 and 749 of the sequence in human C3) of C3:

-   -   P3 P2 P1 P1′ P2′ P3′    -   Leu Ala Arg↓Ser Asn Leu.

Typically, cleavage of a substrate in a biochemical pathway is anactivating cleavage or an inhibitory cleavage. An activating cleavagerefers to cleavage of a polypeptide from an inactive form to an activeform. This includes, for example, cleavage of a zymogen to an activeenzyme. An activating cleavage also is cleavage whereby a protein iscleaved into one or more proteins that themselves have activity. Forexample, the complement system is an irreversible cascade of proteolyticcleavage events whose termination results in the formation of multipleeffector molecules that stimulate inflammation, facilitate antigenphagocytosis, and lyse some cells directly. Thus, cleavage of C3 by a C3convertase into C3a and C3b is an activation cleavage. In contrast, themodified MTSP-1 polypeptides provided herein effect inhibitory cleavageof C3, such as by cleavage in a target site that inactivates C3.

As used herein, an inhibitory cleavage or inactivation cleavage iscleavage of a protein into one or more degradation products that are notfunctional. Inhibitory cleavage results in the diminishment or reductionof an activity of a protein. Typically, a reduction of an activity of aprotein reduces the pathway or process for which the protein isinvolved. In one example, the cleavage of any one or more complementproteins that is an inhibitory cleavage results in the concomitantreduction or inhibition of any one or more of the classical, lectin, oralternative functional pathways of complement. To be inhibitory, thecleavage reduces activity by at least or at least about 1%, 2%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more compared to anative form of the protein. The percent cleavage of a protein that isrequired for the cleavage to be inhibitory varies among proteins but canbe determined by assaying for an activity of the protein.

As used herein, “complement activation” refers to the activation ofcomplement pathways, for example complement activation refers to anincrease in the functions or activities of any one or more of thecomplement pathways by a protease or an increase in the activity of anyof the proteins in the complement pathway. Complement activation canlead to complement-mediated cell lysis or can lead to cell or tissuedestruction. Inappropriate complement activation on host tissue plays animportant role in the pathology of many autoimmune and inflammatorydiseases, and also is responsible for or associated with many diseasestates associated with bioincompatibility. It is understood thatactivation can mean an increase in existing activity as well as theinduction of a new activity. A complement activation can occur in vitroor in vivo. Exemplary functions of complement that can be assayed andthat are described herein include hemolytic assays, and assays tomeasure any one or more of the complement effector molecules such as bySDS PAGE followed by Western Blot or Coomassie Brilliant Blue stainingor by ELISA. In some embodiments, complement activation is inhibited bya protease, such as a protease described herein, by 40%, 50%, 60%, 70%,80%, 85%, 90%, 95% or 99% or more compared to the activity of complementin the absence of a protease.

As used herein, “inhibiting complement activation” or “complementinactivation” refers to the reduction or decrease of acomplement-mediated function or activity of any one or more of thecomplement pathways by a protease or in the activity of any of theproteins in a pathway. A function or activity of complement can occur invitro or in vivo. Exemplary functions of complement that can be assayedand that are described herein include hemolytic assays, and assays tomeasure any one or more of the complement effector molecules such as bySDS PAGE followed by Western Blot or Coomassie Brilliant Blue stainingor by ELISA. A protease can inhibit complement activation by 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In otherembodiments, complement activation is inhibited by a protease by 40%,50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% or more compared to theactivity of complement in the absence of a protease.

As used herein, a “complement protein” or a “complement component” is aprotein of the complement system that functions in the host's defenseagainst infections and in the inflammatory process. Complement proteinsinclude those that function in the classical pathway, those thatfunction in the alternative pathway, and those that function in thelectin pathway. Among the complement proteins are proteases thatparticipate in the complement pathways. In addition, as used herein,complement proteins include any of the “cleavage products” (alsoreferred to as “fragments”) that are formed upon activation of thecomplement cascade. Also included among complement proteins are inactiveor altered forms of complement proteins, such as iC3b and C3a-desArg.Thus, complement proteins include, but are not limited to: C1q, C1r,C1s, C2, C3, C3a, C3b, C3c, C3dg, C3g, C3d, C3f, iC3, C3a-desArg, C4,C4a, C4b, iC4, C4a-desArg, C5, C5a, C5a-des-Arg, C6, C7, C8, C9, MASP-1,MASP-2, MBL, Factor B, Factor D, Factor H, Factor I, CR1, CR2, CR3, CR4,properdin, C1Inh, C4bp, MCP, DAF, CD59 (MIRL), clusterin and HRF andallelic and species variants of any complement protein.

As used herein, a “native” form of a complement protein is one which canbe isolated from an organism such as a vertebrate in the absence ofcomplement activation, and which has not been intentionally modified byman in the laboratory. Examples of native complement proteins includeC1q, C1r, C1s, C2, C3, C4, Factor B, Factor D, properdin, C5, C6, C7,C6, and C9.

Generally, “native complement proteins” are inactive and acquireactivity upon activation. Activation can require activation cleavage,maturation cleavage and/or complex formation with other proteins. Anexception to this is Factor I and Factor D which have enzymatic activityin their native form. In some examples, activation of a nativecomplement protein occurs following cleavage of the protein. Forexample, complement zymogens such as C3 are proteases which arethemselves activated by protease cleavage such that cleavage of C3 bythe C3 convertase C4b2b generates the active fragments C3a and C3b. Inanother example, cleavage of an inactive native complement proteinresults in changes in the structural stability of a protein resulting inactivation of the protein. For example, C3 contains an internalthioester bond which in the native protein is stable, but can becomehighly reactive and activated following conformational changes thatresult from cleavage of the protein. Thus, the cleavage products of C3are biologically active. Activation of C3 also can occur spontaneouslyin the absence of cleavage. It is the spontaneous conversion of thethioester bond in native C3 that is an initiating event of thealternative pathway of complement. In other examples, activation of anative complement protein occurs following the release of a complexedregulatory molecule that inhibits the activity of an otherwise activenative complement protein. For example, C1inh binds to and inactivatesC1s and C1r, unless they are in complex with C1q.

As used herein, “maturation cleavage” is a general term that refers toany cleavage required for activation of a zymogen. This includescleavage that leads to a conformational change resulting in activity(i.e., activation cleavage). It also includes cleavage in which acritical binding site is exposed or a steric hindrance is exposed or aninhibitory segment is removed or moved.

As used herein, “altered form” of a complement protein refers to acomplement protein that is present in a non-native form resulting frommodifications in its molecular structure. For example, C3 reaction ofthe thioester with water can occur in the absence of convertasecleavage, giving a hydrolyzed inactive form of C3 termed iC3. In anotherexample, anaphylatoxins including C3a, C5a, and C4a can be desarginatedby carboxypeptidase N into more stable, less active forms.

As used herein, a “fragment” or “cleavage product” of a complementprotein is a region or segment of a complement protein that contains aportion of the polypeptide sequence of a native complement protein. Afragment of a complement protein usually results following theactivation of a complement cascade. Generally, a fragment results fromthe proteolytic cleavage of a native complement protein. For example,complement protein C3 is enzymatically cleaved by a C3 convertase,resulting in two fragments: C3a which constitutes the N-terminal portionof C3; and C3b which constitutes the C-terminal portion and contains theserine protease site. A fragment of a complement protein also resultsfrom the proteolytic cleavage of another fragment of a complementprotein. For example, C3b, a fragment generated from the cleavage of C3,is cleaved by Factor I to generate the fragments iC3b and C3f. Generallycleavage products of complement proteins are biologically activeproducts and function as cleavage effector molecules of the complementsystem. Hence a fragment or portion of complement protein includescleavage products of complement proteins and also portions of theproteins that retain or exhibit at least one activity of a complementprotein.

As used herein, “cleavage effector molecules” or “cleavage effectorproteins” refers to the active cleavage products generated as a resultof the triggered-enzyme cascade of the complement system. A cleavageeffector molecule, a fragment or a cleavage product resulting fromcomplement activation can contribute to any of one or more of thecomplement-mediated functions or activities, which include opsonization,anaphylaxis, cell lysis and inflammation. Examples of cleavage oreffector molecules include, but are not limited to, C3a, C3b, C4a, C4b,C5a, C5b-9, and Bb. Cleavage effector molecules of the complementsystem, by virtue of participation in the cascade, exhibit activitiesthat include stimulating inflammation, facilitating antigenphagocytosis, and lysing some cells directly. Complement cleavageproducts promote or participate in the activation of the complementpathways.

As used herein, “anaphylatoxins” are cleavage effector proteins thattrigger degranulation of, or release of substances from, mast cells orbasophils, which participate in the inflammatory response, particularlyas part of defense against parasites. If the degranulation is toostrong, it can cause allergic reactions. Anaphylatoxins include, forexample, C3a, C4a and C5a. Anaphylatoxins also indirectly mediate spasmsof smooth muscle cells (such as bronchospasms), increases inpermeability of blood capillaries, and chemotaxis.

As used herein, “chemotaxis” refers to receptor-mediated movement ofleukocytes towards a chemoattractant typically in the direction of theincreasing concentration thereof, such as in the direction of increasingconcentration of an anaphylatoxin.

As used herein, “opsonization” refers to the alteration of the surfaceof a pathogen or other particle so that it can be ingested byphagocytes. A protein that binds or alters the surface of a pathogen istermed an opsonin. Antibody and complement proteins opsonizeextracellular bacteria for uptake and destruction by phagocytes such asneutrophils and macrophages.

As used herein, “cell lysis” refers to the breaking open of a cell bythe destruction of its wall or membrane. Hemolysis of red blood cells isa measure of cell lysis.

As used herein, “complement protein C3” or “C3” refers to complementprotein C3 of the complement system that functions in the host defenseagainst infections and in the inflammatory process. Human complementprotein C3 is a 1663 amino acid single-chain pre-proprotein or zymogenset forth in SEQ ID NO: 9 that that contains a 22 amino acid signalpeptide (amino acids 1-22 of SEQ ID NO: 9) and a tetra-arginine sequence(amino acids 668-671 of SEQ ID NO: 9) that is removed by a furin-likeenzyme resulting in a mature two chain protein containing a beta chain(amino acids 23-667 of SEQ ID NO: 9) and an alpha chain (amino acids672-1663 of SEQ ID NO:9) linked by a disulfide bond between residuesC559 and C816. Complement protein C3 is further activated by proteolyticcleavage by a C3 convertase (C4b2b or C3bBb) between amino acids 748 and749 of SEQ ID NO: 9 generating the anaphylatoxin C3a and the opsoninC3b.

As used herein, a “zymogen” refers to a protein that is activated byproteolytic cleavage, including maturation cleavage, such as activationcleavage, and/or complex formation with other protein(s) and/orcofactor(s). A zymogen is an inactive precursor of a protein. Suchprecursors are generally larger, although not necessarily larger, thanthe active form. With reference to MTSP-1 or complement protein C3,zymogens are converted to active enzymes by specific cleavage, includingcatalytic and autocatalytic cleavage, or by binding of an activatingco-factor, which generates an active enzyme. A zymogen, thus, is anenzymatically inactive protein that is converted to a proteolytic enzymeby the action of an activator. Cleavage can be effectedautocatalytically. A number of complement proteins are zymogens; theyare inactive, but become cleaved and activated upon the initiation ofthe complement system following infection. Zymogens, generally, areinactive and can be converted to mature active polypeptides by catalyticor autocatalytic cleavage of the proregion from the zymogen.

As used herein, a “proregion,” “propeptide,” or “pro sequence,” refersto a region or a segment of a protein that is cleaved to produce amature protein. This can include segments that function to suppressenzymatic activity by masking the catalytic machinery and thuspreventing formation of the catalytic intermediate (i.e., by stericallyoccluding the substrate binding site). A proregion is a sequence ofamino acids positioned at the amino terminus of a mature biologicallyactive polypeptide and can be as little as a few amino acids or can be amultidomain structure.

As used herein, an “activation sequence” refers to a sequence of aminoacids in a zymogen that is the site required for activation cleavage ormaturation cleavage to form an active protease. Cleavage of anactivation sequence can be catalyzed autocatalytically or by activatingpartners.

Activation cleavage is a type of maturation cleavage in which aconformational change required for activity occurs. This is a classicalactivation pathway, for example, for serine proteases in which acleavage generates a new N-terminus which interacts with the conservedregions of catalytic machinery, such as catalytic residues, to induceconformational changes required for activity. Activation can result inproduction of multi-chain forms of the proteases. In some instances,single chain forms of the protease can exhibit proteolytic activity.

As used herein, “domain” refers to a portion of a molecule, such asproteins or the encoding nucleic acids, that is structurally and/orfunctionally distinct from other portions of the molecule and isidentifiable. An exemplary polypeptide domain is a part of thepolypeptide that can form an independently folded structure within apolypeptide made up of one or more structural motifs (e.g., combinationsof alpha helices and/or beta strands connected by loop regions) and/orthat is recognized by a particular functional activity, such asenzymatic activity, dimerization or substrate-binding. A polypeptide canhave one or more, typically more than one, distinct domains. Forexample, the polypeptide can have one or more structural domains and oneor more functional domains. A single polypeptide domain can bedistinguished based on structure and function. A domain can encompass acontiguous linear sequence of amino acids. Alternatively, a domain canencompass a plurality of non-contiguous amino acid portions, which arenon-contiguous along the linear sequence of amino acids of thepolypeptide. Typically, a polypeptide contains a plurality of domains.For example, serine proteases can be characterized based on the sequenceof protease domain(s). Those of skill in the art are familiar withpolypeptide domains and can identify them by virtue of structural and/orfunctional homology with other such domains. For exemplification herein,definitions are provided, but it is understood that it is well withinthe skill in the art to recognize particular domains by name. If needed,appropriate software can be employed to identify domains.

As used herein, a “structural region” of a polypeptide is a region ofthe polypeptide that contains at least one structural domain.

As used herein, a “protease domain” is the catalytically active portionof a protease. Reference to a protease domain of a protease includes thesingle, two- and multi-chain forms of any of these proteins. A proteasedomain of a protein contains all of the requisite properties of thatprotein required for its proteolytic activity, such as for example, itscatalytic center.

As used herein, a “catalytically active portion” or “catalyticallyactive domain” of a protease, for example an MTSP-1 polypeptide, refersto the protease domain, or any fragment or portion thereof that retainsprotease activity. For example, a catalytically active portion of aMTSP-1 polypeptide can be a MTSP-1 protease domain including an isolatedsingle chain form of the protease domain or an activated two-chain form.The zymogen form of each protein is single chain form, which can beconverted to the active two chain form by cleavage. The protease domainalso can be converted to a two chain form. Significantly, at least invitro, the single chain forms of the proteases and catalytic domains orproteolytically active portions thereof (typically C-terminaltruncations) exhibit protease activity.

As used herein, a “nucleic acid encoding a protease domain orcatalytically active portion of a protease” refers to a nucleic acidencoding only the recited single chain protease domain or active portionthereof, and not the other contiguous portions of the protease as acontinuous sequence.

As used herein, recitation that a polypeptide consists essentially ofthe protease domain means that the only portion of the polypeptide is aprotease domain or a catalytically active portion thereof. Thepolypeptide can optionally, and generally will, include additionalnon-protease-derived sequences of amino acids.

As used herein, an “active site of a protease” refers to the substratebinding site where catalysis of the substrate occurs. The structure andchemical properties of the active site allow the recognition and bindingof the substrate and subsequent hydrolysis and cleavage of the scissilebond in the substrate. The active site of a protease contains aminoacids that contribute to the catalytic mechanism of peptide cleavage, aswell as amino acids that contribute to substrate sequence recognition,such as amino acids that contribute to extended substrate bindingspecificity.

As used herein, target site in C3 refers to a site that, when cleaved,inactivates C3. Exemplary of such site is:

-   -   Q H A R↓A S H L (residues 737-744 of SEQ ID NO:9)    -   P4P3P2P1↓P1′ P4′.

As used herein, the “substrate recognition site” or “cleavage sequence”refers to the sequence recognized by the active site of a protease thatis cleaved by a protease. Typically, a cleavage sequence for a serineprotease is six residues in length to match the extended substratespecificity of many proteases, but can be longer or shorter dependingupon the protease. Typically, for example, for a serine protease, acleavage sequence is made up of the P1-P4 and P1′-P4′ amino acids in asubstrate, where cleavage occurs after the P1 position. Typically, acleavage sequence for a serine protease is six residues in length tomatch the extended substrate specificity of many proteases, but can belonger or shorter depending upon the protease.

As used herein, “target substrate” refers to a substrate that is cleavedby a protease. Typically, the target substrate is specifically cleavedat its substrate recognition site by a protease. Minimally, a targetsubstrate includes the amino acids that make up the cleavage sequence.Optionally, a target substrate includes a peptide containing thecleavage sequence and any other amino acids. A full-length protein,allelic variant, isoform, or any portion thereof, containing a cleavagesequence recognized by a protease, is a target substrate for thatprotease. For example, for purposes herein in which complementinactivation is intended, a target substrate is complement protein C3,or any portion or fragment thereof containing a cleavage sequencerecognized by a MTSP-1 polypeptide. Such target substrates can bepurified proteins, or can be present in a mixture, such as a mixture invitro or a mixture in vivo. Mixtures can include, for example, blood orserum or breast milk, or other tissue fluids. Additionally, a targetsubstrate includes a peptide or protein containing an additional moietythat does not affect cleavage of the substrate by a protease. Forexample, a target substrate can include a four amino acid peptide or afull-length protein chemically linked to a fluorogenic moiety. Theproteases can be modified to exhibit greater substrate specificity for atarget substrate.

As used herein, “MTSP-1” or “MTSP1” or “membrane-type serine protease”“MTSP-1 polypeptide” refers to any MTSP-1 polypeptide including, but notlimited to, a recombinantly produced polypeptide, a syntheticallyproduced polypeptide and a MTSP-1 polypeptide extracted or isolated fromcells or tissues including, but not limited to, epithelial cells, cancercells, liver and blood. Alternative names that are used interchangeablyfor MTSP-1 include membrane-type serine protease and Matriptase andEpithin and TMPRSS1 and Suppressor of tumorigenicity 14 protein andProstamin and Serine protease 14 (ST14) and Serine protease TADG-15 andTumor-associated differentially-expressed gene 15 (TADG-15) protein.MTSP-1 includes related polypeptides from different species including,but not limited to animals of human and non-human origin. Human MTSP-1includes MTSP-1, allelic variants, isoforms, synthetic molecules fromnucleic acids, protein isolated from human tissue and cells, andmodified forms thereof.

Exemplary unmodified human MTSP-1 polypeptides include, but are notlimited to, unmodified and wild-type MTSP-1 polypeptides (SEQ ID NO:1)and the protease domain (such as the single-chain protease domain ofMTSP-1 set forth in SEQ ID NO: 2). One of skill in the art wouldrecognize that the referenced positions of the full length wild-typeMTSP-1 polypeptide (SEQ ID NO:1) differ by 614 amino acid residues whencompared to the MTSP-1 protease domain (SEQ ID NO:2). Thus, the firstamino acid residue of SEQ ID NO:2 “corresponds to” the six hundred andfifteenth (615th) amino acid residue of SEQ ID NO:1. In anotherembodiment, an MTSP-1 polypeptide can be any one or more of the allelicvariants of MTSP-1 as set forth herein.

An MTSP-1 protease occurs as a single chain zymogen, and as an activatedtwo-chain polypeptide. Reference to MTSP-1 includes active single-chainand two-chain forms thereof. The MTSP-1 polypeptides provided herein canbe further modified, such as by chemical modification orpost-translational modification. Such modifications include, but are notlimited to, glycosylation, PEGylation, albumination, farnesylation,carboxylation, hydroxylation, phosphorylation, HESylation, PASylation,and other polypeptide modifications known in the art.

MTSP-1 includes MTSP-1 from any species, including human and non-humanspecies. MTSP-1 polypeptides of non-human origin include, but are notlimited to, murine and rat MTSP-1 polypeptides. Exemplary MTSP-1polypeptides of non-human origin include, for example, mouse (Musmusculus, SEQ ID NO:12) and rat (Rattus norvegicus, SEQ ID NO:13).

Reference to MTSP-1 polypeptides also includes precursor polypeptidesand mature MTSP-1 polypeptides in single-chain or two-chain forms,truncated forms thereof that have activity, the isolated protease domainand includes allelic variants and species variants, variants encoded bysplice variants, and other variants, including polypeptides that have atleast or at least about 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to full lengthform (SEQ ID NO:1 or 3) or the protease domain thereof (SEQ ID NO: 2 or4). MTSP-1 polypeptides include, but are not limited to, tissue-specificisoforms and allelic variants thereof, synthetic molecules prepared bytranslation of nucleic acids, proteins generated by chemical synthesis,such as syntheses that include ligation of shorter polypeptides, throughrecombinant methods, proteins isolated from human and non-human tissueand cells, chimeric MTSP-1 polypeptides, and modified forms thereof.MTSP-1 polypeptides also include fragments or portions of MTSP-1 thatare of sufficient length or include appropriate regions to retain atleast one activity (upon activation if needed) of a full-length maturepolypeptide. In one example the portion of MTSP-1 is the proteasedomain, such as, for example, the protease domain set forth in SEQ IDNO: 2 which corresponds to amino acids 615-855 of the WT MTSP-1 sequenceset forth in SEQ ID NO: 1. MTSP-1 polypeptides also include those thatcontain chemical or posttranslational modifications and those that donot contain chemical or posttranslational modifications. Suchmodifications include, but are not limited to, PEGylation, albumination,glycosylation, farnesylation, carboxylation, hydroxylation,phosphorylation, HESylation, PASylation, and other polypeptidemodifications known in the art.

As used herein, “MTSP-1 protease” or “MTSP-1 protease domain” refers toany MTSP-1 polypeptide including, but not limited to, a recombinantlyproduced polypeptide, a synthetically produced polypeptide and a MTSP-1polypeptide extracted or isolated from cells or tissues including, butnot limited to, liver and blood. MTSP-1 protease includes relatedpolypeptides from different species including, but not limited toanimals of human and non-human origin. A human MTSP-1 protease or MTSP-1protease domain includes MTSP-1, allelic variants, isoforms, syntheticmolecules from nucleic acids, protein isolated from human tissue andcells, and modified forms thereof. Exemplary reference human MTSP-1protease domains include, but are not limited to, unmodified andwild-type MTSP-1 protease domain (SEQ ID NO:2) and an alternate proteasedomain (such as the MTSP-1 protease domain set forth in SEQ ID NO: 4).One of skill in the art would recognize that the referenced positions ofthe MTSP-1 protease domain (SEQ ID NO:2) differ by 614 amino acidresidues when compared to the full length MTSP-1 polypeptide (SEQ IDNO:1), which is the MTSP-1 polypeptide containing the full length WTsequence. Thus, the first amino acid residue of SEQ ID NO:2 “correspondsto” the six hundred and fifteenth (615th) amino acid residue of SEQ IDNO:1.

As used herein, a “modification” is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids or nucleotides, respectively. ModifiedMT-SP1 polypeptides refer to MT-SP1 polypeptides containing alterationsin the primary sequence of the polypeptide. Methods of modifying apolypeptide are routine to those of skill in the art, such as by usingrecombinant DNA methodologies. Reference to other modifications, such aspost-translational modifications, and conjugation to moieties, such aspolymers, such as PEG moieties, and tags for detection or isolation, arespecified as such.

As used herein, “substitution” or “replacement” refers to the replacingof one or more nucleotides or amino acids in a native, target, wild-typeor other nucleic acid or polypeptide sequence with an alternativenucleotide or amino acid, without changing the length (as described innumbers of residues) of the molecule. Thus, one or more substitutions ina molecule does not change the number of amino acid residues ornucleotides of the molecule. Amino acid replacements compared to aparticular polypeptide can be expressed in terms of the number of theamino acid residue along the length of the polypeptide sequence. Forexample, a modified polypeptide having a modification in the amino acidat the 35^(th) position of the amino acid sequence that is asubstitution/replacement of Arginine (Arg; R) with glutamine (Gln; Q)can be expressed as R35Q, Arg35G1n, or 35Q. Simply R35 can be used toindicate that the amino acid at the modified 35^(th) position is anarginine.

As used herein, a “modified MTSP-1” or “modified MTSP-1 polypeptide”refers to a MTSP-1 protease that exhibits altered activity, such asaltered substrate specificity, compared to the unmodified form. Suchproteases include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more modifications (i.e., changes in amino acids)compared to a wild type MTSP-1 such that an activity, such as substratespecificity or selectivity, of the MTSP-1 protease for cleavingcomplement protein C3 is altered. A modified MTSP-1 can be a full-lengthMTSP-1 protease, or can be a portion thereof of a full length protease,such as the protease domain of MTSP-1, as long as the modified MTSP-1protease contains modifications in regions that alter the activity orsubstrate specificity of the protease and the protease isproteolytically active. A modified MTSP-1 protease, or a modified MTSP-1protease domain, also can include other modifications in regions that donot impact on substrate specificity of the protease. Hence, a modifiedMTSP-1 polypeptide typically has 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to acorresponding sequence of amino acids of a wild type MTSP-1 polypeptide.A modified full-length MTSP-1 polypeptide or a catalytically activeportion thereof or a protease domain thereof of a modified MTSP-1polypeptide can include polypeptides that are fusion proteins as long asthe fusion protein possesses the target specificity.

As used herein, chymotrypsin numbering refers to the amino acidnumbering of a mature chymotrypsin polypeptide, corresponding toresidues 19-263 of SEQ ID NO:14. Alignment of a protease domain ofanother protease, such as for example, the protease domain of MTSP-1,can be made with chymotrypsin. In such an instance, the amino acids ofMTSP-1 that correspond to amino acids of chymotrypsin are given thenumbering of the chymotrypsin amino acids. Corresponding positions canbe determined by such alignment by one of skill in the art using manualalignments or by using the numerous alignment programs available (forexample, BLASTP). Corresponding positions also can be based onstructural alignments, for example by using computer simulatedalignments of protein structure. Recitation that amino acids of apolypeptide correspond to amino acids in a disclosed sequence refers toamino acids identified upon alignment of the polypeptide with thedisclosed sequence to maximize identity or homology (where conservedamino acids are aligned) using a standard alignment algorithm, such asthe GAP algorithm. The corresponding chymotrypsin numbers of amino acidpositions 615-855 of the MTSP-1 polypeptide set forth in SEQ ID NO:1 areprovided in Table 1. The amino acid positions relative to the sequenceset forth in SEQ ID NO:1 are in normal font, the amino acid residues atthose positions are in bold, and the corresponding chymotrypsin numbersare in italics. For example, upon alignment of the serine proteasedomain of MTSP-1 (SEQ ID NO: 2) with mature chymotrypsin, V at position1 in the MTSP-1 protease domain is given the chymotrypsin numbering ofV16. Subsequent amino acids are numbered accordingly. In one example, anF at amino acid position 708 of full-length MTSP-1 (SEQ ID NO:1) or atposition 94 of the protease domain of MTSP-1 (SEQ ID NO:2), correspondsto F99 based on chymotrypsin numbering. Where a residue exists in aprotease, but is not present in chymotrypsin, the amino acid residue isgiven a letter notation. For example, residues in chymotrypsin that arepart of a loop with amino acid 60 based on chymotrypsin numbering, butare inserted in the MTSP-1 sequence compared to chymotrypsin, arereferred to for example as D60b or R60c. These residues correspond toD661 and R662, respectively, by numbering relative to the mature MTSP-1sequence (human) set forth in SEQ ID NO:1.

TABLE 1 Chymotrypsin numbering of MTSP-1 615 616 617 618 619 620 621 622623 624 625 626 627 628 629 V V G G T D A D E G E W P W Q 16 17 18 19 2021 22 23 24 25 26 27 28 29 30 630 631 632 633 634 635 636 637 638 639640 641 642 643 644 V S L H A L G Q G H I C G A S 31 32 33 34 35 36 3738 39 40 41 42 43 44 45 645 646 647 648 649 650 651 652 653 654 655 656657 658 659 L I S P N W L V S A A H C Y I 46 47 48 49 50 51 52 53 54 5556 57 58 59 60 660 661 662 663 664 665 666 667 668 669 670 671 672 673674 D D R G F R Y S D P T Q W T A 60a 60b 60c 60d 60e 60f 60g 60h 601 6162 63 64 65 66 675 676 677 678 679 680 681 682 683 684 685 686 687 688689 F L G L H D Q S Q R S A P G V 67 68 69 70 71 72 73 74 74a 75 76 7778 79 80 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 Q ER R L K R I I S H P F F N 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95705 706 707 708 709 710 711 712 713 714 715 716 717 718 D F T F D Y D IA L L E L E 96 97 97a 98 99 100 101 102 103 104 105 106 107 108 109 719720 721 722 723 724 725 726 727 728 729 730 731 732 733 K P A E Y S S MV R P I C L P 110 111 112 113 114 115 116 117 118 119 120 121 122 123124 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 D A S HV F P A G K A I W V T 125 126 127 128 129 130 131 132 133 134 135 136137 138 139 749 750 751 752 753 754 755 756 757 758 759 760 761 762 G WG H T Q Y G G T G A L I 140 141 142 143 144 145 146 147 148 149 150 151152 153 154 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777L Q K G E I R V I N Q T T C E 155 156 157 158 159 160 161 162 163 164165 166 167 168 169 778 779 780 781 782 783 784 785 786 787 788 789 790791 792 N L L P Q Q I T P R M M C V G 170 171 172 173 174 175 176 177178 179 180 181 182 183 184 793 794 795 796 797 798 799 800 801 802 803804 805 806 807 F L S G G V D S C Q G D S G G 184a 185 186 186a 187 188189 190 191 192 193 194 195 196 197 808 809 810 811 812 813 814 815 816817 818 819 820 821 822 P L S S V E A D G R I F Q A G 198 199 200 201202 203 204 204a 205 206 207 208 209 210 211 823 824 825 826 827 828 829830 831 832 833 834 835 836 837 V V S W G D G C A Q R N K P G 212 213214 215 216 217 218 219 220 221 222 223 224 225 226 838 839 840 841 842843 844 845 846 847 848 849 850 851 852 V Y T R L P L F R D W I K E N227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 853 854 855T G V 242 243 244

As used herein, k_(cat) is a measure of the catalytic activity of anenzyme; the units of k_(cat) are seconds⁻¹. The reciprocal of k_(cat) isthe time required by an enzyme molecule to “turn over” one substratemolecule; k_(cat) measures the number of substrate molecules turned overper enzyme molecule per second. k_(cat) also is called the turnovernumber.

As used herein, specificity for a target substrate refers to apreference for cleavage of a target substrate by a protease compared toa another substrate, referred to as a non-target substrate. Specificityis reflected in the specificity constant (k_(cat)/K_(m)), which is ameasure of the affinity of a protease for its substrate and theefficiency of the enzyme. k_(cat)/K_(m) is a measure of enzymeefficiency; a large value of k_(cat) (rapid turnover) or a small valueof K_(m) (high affinity for substrate) makes k_(cat)/K_(m) large.

As used herein, a specificity constant for cleavage is (k_(cat)/K_(m)),wherein K_(m) is the Michaelis-Menton constant ([S] at one half V_(max))and k_(cat) is the V_(max)/[E_(T)], where E_(T) is the final enzymeconcentration. The parameters k_(cat), K_(m) and k_(cat)/K_(m) can becalculated by graphing the inverse of the substrate concentration versusthe inverse of the velocity of substrate cleavage, and fitting to theLineweaver-Burk equation (1/velocity=(K_(m)/V_(max))(1/[S])+1/V_(max);where V_(max)=[E_(T)]k_(cat)). Any method to determine the rate ofincrease of cleavage over time in the presence of various concentrationsof substrate can be used to calculate the specificity constant. Forexample, a substrate is linked to a fluorogenic moiety, which isreleased upon cleavage by a protease. By determining the rate ofcleavage at different enzyme concentrations, k_(cat) can be determinedfor a particular protease. The specificity constant can be used todetermine the preference of a protease for one target substrate overanother substrate.

As used herein, substrate specificity refers to the preference of aprotease for one target substrate over another. Substrate specificitycan be measured as a ratio of specificity constants.

As used herein, a substrate specificity ratio is the ratio ofspecificity constants and can be used to compare specificities of two ormore proteases or a protease for two more substrates. For example,substrate specificity of a protease for competing substrates or ofcompeting proteases for a substrate can be compared by comparingk_(cat)/K_(m). For example, a protease that has a specificity constantof 2×10⁶ M⁻¹ sec⁻¹ for a target substrate and 2×10⁴ M⁻¹ sec⁻¹ for anon-target substrate is more specific for the target substrate. Usingthe specificity constants from above, the protease has a substratespecificity ratio of 100 for the target substrate.

As used herein, preference or substrate specificity for a targetsubstrate can be expressed as a substrate specificity ratio. Theparticular value of the ratio that reflects a preference is a functionof the substrates and proteases at issue. A substrate specificity ratiothat is greater than 1 signifies a preference for a target substrate anda substrate specificity less than 1 signifies a preference for anon-target substrate. Generally, a ratio of at least or about 1 reflectsa sufficient difference for a protease to be considered a candidatetherapeutic.

As used herein, altered specificity refers to a change in substratespecificity of a modified protease compared to a starting wild typeprotease. Generally, the change in specificity is a reflection of thechange in preference of a modified protease for a target substratecompared to a wild type substrate of the protease (herein referred to asa non-target substrate). Typically, modified MTSP-1 proteases providedherein exhibit increased substrate specificity for complement protein C3compared to the substrate specificity of the wild type MTSP-1 protease.For example, a modified protease that has a substrate specificity ratioof 100 for a target substrate versus a non-target substrate exhibits a10-fold increased specificity compared to a scaffold protease with asubstrate specificity ratio of 10. In another example, a modifiedprotease that has a substrate specificity ratio of 1 compared to a ratioof 0.1, exhibits a 10-fold increase in substrate specificity. To exhibitincreased specificity compared to a scaffold protease, a modifiedprotease has a 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold,200-fold, 300-fold, 400-fold, 500-fold or more greater substratespecificity for any one of more of the complement proteins.

As used herein, “selectivity” can be used interchangeably withspecificity when referring to the ability of a protease to choose andcleave one target substrate from among a mixture of competingsubstrates. Increased selectivity of a protease for a target substratecompared to any other one or more target substrates can be determined,for example, by comparing the specificity constants of cleavage of thetarget substrates by a protease. For example, if a protease has aspecificity constant of cleavage of 2×10⁶ M⁻¹ sec⁻¹ for a targetsubstrate and 2×10⁴ M⁻¹ sec⁻¹ for any other one of more substrates, theprotease is more selective for the target substrate.

As used herein, an “activity” or a “functional activity” of apolypeptide, such as a protease, refers to any activity exhibited by thepolypeptide. Such activities can be empirically determined. Exemplaryactivities include, but are not limited to, ability to interact with abiomolecule, for example, through substrate-binding, DNA binding, ordimerization, and enzymatic activity, for example, kinase activity orproteolytic activity. For a protease (including protease fragments),activities include, but are not limited to, the ability to specificallybind a particular substrate, affinity and/or specificity ofsubstrate-binding (e.g., high or low affinity and/or specificity),effector functions, such as the ability to promote substrate (e.g.,protein, i.e. C3) inhibition, neutralization, cleavage or clearance, andin vivo activities, such as the ability to promote protein cleavage orclearance. Activity can be assessed in vitro or in vivo using recognizedassays, such as ELISA, flow cytometry, surface plasmon resonance orequivalent assays to measure on- or off-rate, immunohistochemistry andimmunofluorescence histology and microscopy, cell-based assays, andbinding assays. For example, for a protease, e.g., a modified MTSP-1protease, activities can be assessed by measuring substrate proteincleavage, turnover, residual activity, stability and/or levels in vitroand/or in vivo. The results of such in vitro assays that indicate that apolypeptide exhibits an activity can be correlated to activity of thepolypeptide in vivo, in which in vivo activity can be referred to astherapeutic activity, or biological activity. Activity of a modifiedpolypeptide can be any level of percentage of activity of the unmodifiedpolypeptide, including, but not limited to, at or about 1% of theactivity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%,500%, or more of activity compared to the unmodified polypeptide. Assaysto determine functionality or activity of modified (or variant)proteases are well-known in the art.

Functional activities include, but are not limited to, biologicalactivity, catalytic or enzymatic activity, antigenicity (ability to bindto or compete with a polypeptide for binding to an anti-polypeptideantibody), immunogenicity, ability to form multimers, and the ability tospecifically bind to a receptor or ligand for the polypeptide.

As used herein, a functional activity with reference to a complementprotein refers to a complement-mediated function including, but notlimited to, anaphylaxis, opsonization, chemotaxis, or cell lysis.Non-limiting assays for testing activities of complement includehemolysis of red blood cells, and detection of complement effectormolecules such as by ELISA or SDS-PAGE.

As used herein, catalytic activity or cleavage activity refers to theactivity of a protease as assessed in in vitro proteolytic assays thatdetect proteolysis of a selected substrate. Cleavage activity can bemeasured by assessing catalytic efficiency of a protease.

As used herein, activity towards a target substrate refers to cleavageactivity and/or functional activity, or other measurement that reflectsthe activity of a protease on or towards a target substrate. Afunctional activity of a complement protein target substrate by aprotease can be measured by assessing an IC₅₀ in a complement assay suchas red blood cell lysis, or other such assays known by one of skill inthe art or provided herein to assess complement activity. Cleavageactivity can be measured by assessing catalytic efficiency of aprotease. For purposes herein, an activity is increased if a proteaseexhibits greater proteolysis or cleavage of a target substrate and/ormodulates (i.e., activates or inhibits) a functional activity of acomplement protein as compared to in the absence of the protease.

As used herein, “increased activity” with reference to a modified MTSP-1polypeptide means that, when tested under the same conditions, themodified MTSP-1 polypeptide exhibits greater activity compared to anunmodified MTSP-1 polypeptide not containing the amino acidreplacement(s). For example, a modified MTSP-1 polypeptide exhibits atleast or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% ormore of the activity of the unmodified or reference MTSP-1 polypeptide.

As used herein, the term “the same,” when used in reference to antibodybinding affinity, means that the EC₅₀, association constant (Ka) ordissociation constant (Kd) is within about 1 to 100 fold or 1 to 10 foldof that of the reference antibody (1-100 fold greater affinity or 1-100fold less affinity, or any numerical value or range or value within suchranges, than the reference antibody).

As used herein, binding activity refers to characteristics of amolecule, e.g., a polypeptide, relating to whether or not, and how, itbinds one or more binding partners. Binding activities include theability to bind the binding partner(s), the affinity with which it bindsto the binding partner (e.g., high affinity), the strength of the bondwith the binding partner and/or specificity for binding with the bindingpartner.

As used herein, EC₅₀, also called the apparent Kd, is the concentration(e.g., nM) of protease, where 50% of the maximal activity is observed ona fixed amount of substrate (e.g., the concentration of modified MTSP-1polypeptide required to cleave through 50% of the available hC3).Typically, EC₅₀ values are determined from sigmoidal dose-responsecurves, where the EC₅₀ is the concentration at the inflection point. Ahigh protease affinity for its substrate correlates with a low EC₅₀value and a low affinity corresponds to a high EC₅₀ value. Affinityconstants can be determined by standard kinetic methodology for proteasereactions, for example, immunoassays, such as ELISA, followed bycurve-fitting analysis.

As used herein, “affinity constant” refers to an association constant(Ka) used to measure the affinity or molecular binding strength betweena protease and a substrate. The higher the affinity constant the greaterthe affinity of the protease for the substrate. Affinity constants areexpressed in units of reciprocal molarity (i.e., M⁻¹) and can becalculated from the rate constant for the association-dissociationreaction as measured by standard kinetic methodology forprotease-substrate reactions (e.g., immunoassays, surface plasmonresonance, or other kinetic interaction assays known in the art). Thebinding affinity of an protease also can be expressed as a dissociationconstant, or Kd. The dissociation constant is the reciprocal of theassociation constant, Kd=1/Ka. Hence, an affinity constant also can berepresented by the Kd. Affinity constants can be determined by standardkinetic methodology for protease reactions, for example, immunoassays,surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin.Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermaltitration calorimetry (ITC) or other kinetic interaction assays known inthe art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., RavenPress, New York, pages 332-336 (1989)). Instrumentation and methods forreal time detection and monitoring of binding rates are known and arecommercially available (e.g., BIAcore 2000, BIAcore AB, Uppsala, Swedenand GE Healthcare Life Sciences; Malmqvist (1999) Biochem. Soc. Trans.27:335).

Methods for calculating affinity are well-known, such as methods fordetermining EC₅₀ values or methods for determiningassociation/dissociation constants. For example, in terms of EC₅₀, highbinding affinity means that the protease specifically binds to a targetprotein with an EC₅₀ that is less than about 10 ng/mL, 9 ng/mL, 8 ng/mL,7 ng/mL, 6 ng/mL, 5 ng/mL, 4 ng/mL, 3 ng/mL, 2 ng/mL, 1 ng/mL or less.High binding affinity also can be characterized by an equilibriumdissociation constant (Kd) of 10⁻⁶ M or lower, such as 10⁻⁷M, 10⁻⁸M,10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M or lower. In terms of equilibriumassociation constant (Ka), high binding affinity is generally associatedwith Ka values of greater than or equal to about 10⁶ M⁻¹, greater thanor equal to about 10⁷ M⁻¹, greater than or equal to about 10⁸ M⁻¹, orgreater than or equal to about 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹ or 10¹² M⁻¹.Affinity can be estimated empirically or affinities can be determinedcomparatively, e.g., by comparing the affinity of two or more antibodiesfor a particular antigen, for example, by calculating pairwise ratios ofthe affinities of the antibodies tested. For example, such affinitiescan be readily determined using conventional techniques, such as byELISA; equilibrium dialysis; surface plasmon resonance; byradioimmunoassay using radiolabeled target antigen; or by another methodknown to the skilled artisan. The affinity data can be analyzed, forexample, by the method of Scatchard et al., Ann N.Y. Acad. Sci., 51:660(1949) or by curve fitting analysis, for example, using a 4 ParameterLogistic nonlinear regression model using the equation:y=((A−D)/(1+((x/C){circumflex over ( )}B)))+D, where A is the minimumasymptote, B is the slope factor, C is the inflection point (EC₅₀), andD is the maximum asymptote.

As used herein, “ED₅₀” is the dose (e.g., mg/kg or nM) of a protease(e.g., a modified MTSP-1 protease) that produces a specified result(e.g., cleavage of the complement protein C3) in 50% of the totalpopulation (e.g., total amount of C3 present in the sample).

As used herein, “substantially the same” when used in reference to EC₅₀,association constant (Ka) or dissociation constant (Kd), or ED₅₀effective dose means that the Ka, Kd, EC₅₀ or ED₅₀ is within about 5 to5000 fold greater or less than the Ka, Kd, EC₅₀ or ED₅₀, of thereference MTSP-1 (5-5000 fold greater or 5-5000 fold less than thereference MTSP-1, i.e., wild-type MTSP-1).

As used herein, the term “surface plasmon resonance” refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example, using the BIAcore system (GEHealthcare Life Sciences).

As used herein, a human protein is one encoded by a nucleic acidmolecule, such as DNA, present in the genome of a human, including allallelic variants and conservative variations thereof. A variant ormodification of a protein is a human protein if the modification isbased on the wild type or prominent sequence of a human protein.

As used herein, the residues of naturally occurring α-amino acids arethe residues of those 20 α-amino acids found in nature which areincorporated into protein by the specific recognition of the chargedtRNA molecule with its cognate mRNA codon in humans.

As used herein, non-naturally occurring amino acids refer to amino acidsthat are not genetically encoded.

As used herein, “nucleic acid” refers to at least two linked nucleotidesor nucleotide derivatives, including a deoxyribonucleic acid (DNA) and aribonucleic acid (RNA) and analogs thereof, joined together, typicallyby phosphodiester linkages. Also included in the term “nucleic acid” areanalogs of nucleic acids such as peptide nucleic acid (PNA),phosphorothioate DNA, and other such analogs and derivatives orcombinations thereof. Nucleic acids also include DNA and RNA derivativescontaining, for example, a nucleotide analog or a “backbone” bond otherthan a phosphodiester bond, for example, a phosphotriester bond, aphosphoramidate bond, a phosphorothioate bond, a thioester bond, or apeptide bond (peptide nucleic acid). The term also includes, asequivalents, derivatives, variants and analogs of either RNA or DNA madefrom nucleotide analogs, single (sense or antisense) and double-strandednucleic acids. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracilbase is uridine. Nucleic acids can be single or double-stranded. Whenreferring to probes or primers, which are optionally labeled, such aswith a detectable label, such as a fluorescent or radiolabel,single-stranded molecules are contemplated. Such molecules are typicallyof a length such that their target is statistically unique or of lowcopy number (typically less than 5, generally less than 3) for probingor priming a library. Generally a probe or primer contains at least 14,16 or 30 contiguous nucleotides of sequence complementary to oridentical to a gene of interest. Probes and primers can be 10, 20, 30,50, 100 or more nucleotides long.

As used herein, an isolated nucleic acid molecule is one which isseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid molecule. An “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Exemplary isolated nucleic acidmolecules provided herein include isolated nucleic acid moleculesencoding a MTSP-1 protease provided.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, “polypeptide” refers to two or more amino acidscovalently joined. The terms “polypeptide” and “protein” are usedinterchangeably herein.

As used herein, a “peptide” refers to a polypeptide that is from 2 toabout or 40 amino acids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 2). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids (Table 2), non-natural amino acids andamino acid analogs (i.e., amino acids wherein the α-carbon has a sidechain).

As used herein, the amino acids, which occur in the various amino acidsequences of polypeptides herein, are identified according to theirwell-known, three-letter or one-letter abbreviations (see Table 2). Thenucleotides, which occur in the various nucleic acid molecules andfragments, are designated with the standard single-letter designationsused routinely in the art.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted in 37 C.F.R. §§ 1.821-1.822, abbreviations for amino acidresidues are shown in Table 2:

TABLE 2 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

All sequences of amino acid residues represented herein by a formulahave a left to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is defined to include the amino acids listed in the Table ofCorrespondence (Table 2), modified, non-natural and unusual amino acids.Furthermore, a dash at the beginning or end of an amino acid residuesequence indicates a peptide bond to a further sequence of one or moreamino acid residues or to an amino-terminal group such as NH₂ or to acarboxyl-terminal group such as COOH.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides. As used herein, the residuesof naturally occurring α-amino acids are the residues of those 20α-amino acids found in nature which are incorporated into protein by thespecific recognition of the charged tRNA molecule with its cognate mRNAcodon in humans.

As used herein, “non-natural amino acid” refers to an organic compoundthat has a structure similar to a natural amino acid but has beenmodified structurally to mimic the structure and reactivity of a naturalamino acid. Non-naturally occurring amino acids, thus, include, forexample, amino acids or analogs of amino acids other than the 20naturally occurring amino acids and include, but are not limited to, theD-stereoisomers of amino acids. Exemplary non-natural amino acids areknown to those of skill in the art, and include, but are not limited to,para-acetyl Phenylalanine, para-azido Phenylalanine, 2-Aminoadipic acid(Aad), 3-Aminoadipic acid (bAad), β-alanine/β-Amino-propionic acid(Bala), 2-Aminobutyric acid (Abu), 4-Aminobutyric acid/piperidinic acid(4Abu), 6-Aminocaproic acid (Acp), 2-Aminoheptanoic acid (Ahe),2-Aminoisobutyric acid (Aib), 3-Aminoisobutyric acid (Baib),2-Aminopimelic acid (Apm), 2,4-Diaminobutyric acid (Dbu), Desmosine(Des), 2,2′-Diaminopimelic acid (Dpm), 2,3-Diaminopropionic acid (Dpr),N-Ethylglycine (EtGly), N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl),allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline(4Hyp), Isodesmosine (Ide), allo-Isoleucine (Aile), N-Methylglycine,sarcosine (MeGly), N-Methylisoleucine (MeIle), 6-N-Methyllysine (MeLys),N-Methylvaline (MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine(Orn). Exemplary non-natural amino acids are described herein and areknown to those of skill in the art.

As used herein, an isokinetic mixture is one in which the molar ratiosof amino acids has been adjusted based on their reported reaction rates(see, e.g., Ostresh et al. (1994) Biopolymers 34:1681).

As used herein, a DNA construct is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term ortholog means a polypeptide or proteinobtained from one species that is the functional counterpart of apolypeptide or protein from a different species. Sequence differencesamong orthologs are the result of speciation.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule cannot be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, alignment of a sequence refers to the use of homology toalign two or more sequences of nucleotides or amino acids. Typically,two or more sequences that are related by 50% or more identity arealigned. An aligned set of sequences refers to 2 or more sequences thatare aligned at corresponding positions and can include aligningsequences derived from RNAs, such as ESTs and other cDNAs, aligned withgenomic DNA sequences. Related or variant polypeptides or nucleic acidmolecules can be aligned by any method known to those of skill in theart. Such methods typically maximize matches, and include methods, suchas using manual alignments and by using the numerous alignment programsavailable (e.g., BLASTP) and others known to those of skill in the art.By aligning the sequences of polypeptides or nucleic acids, one skilledin the art can identify analogous portions or positions, using conservedand identical amino acid residues as guides. Further, one skilled in theart also can employ conserved amino acid or nucleotide residues asguides to find corresponding amino acid or nucleotide residues betweenand among human and non-human sequences. Corresponding positions alsocan be based on structural alignments, for example by using computersimulated alignments of protein structure. In other instances,corresponding regions can be identified. One skilled in the art also canemploy conserved amino acid residues as guides to find correspondingamino acid residues between and among human and non-human sequences.

As used herein, “sequence identity” refers to the number of identical orsimilar amino acids or nucleotide bases in a comparison between a testand a reference polypeptide or polynucleotide. Sequence identity can bedetermined by sequence alignment of nucleic acid or protein sequences toidentify regions of similarity or identity. For purposes herein,sequence identity is generally determined by alignment to identifyidentical residues. The alignment can be local or global. Matches,mismatches and gaps can be identified between compared sequences. Gapsare null amino acids or nucleotides inserted between the residues ofaligned sequences so that identical or similar characters are aligned.Generally, there can be internal and terminal gaps. Sequence identitycan be determined by taking into account gaps as the number of identicalresidues/length of the shortest sequence×100. When using gap penalties,sequence identity can be determined with no penalty for end gaps (e.g.,terminal gaps are not penalized). Alternatively, sequence identity canbe determined without taking into account gaps as the number ofidentical positions/length of the total aligned sequence×100.

As used herein, “at a position corresponding to” or recitation thatnucleotides or amino acid positions “correspond to” nucleotides or aminoacid positions in a disclosed sequence, such as set forth in theSequence listing, refers to nucleotides or amino acid positionsidentified upon alignment with the disclosed sequence to maximizeidentity using a standard alignment algorithm, such as the GAPalgorithm. For purposes herein, alignment of a MTSP-1 sequence is to theamino acid sequence of the protease domain of human MTSP-1 set forth inSEQ ID NO: 2, or particularly a reference MTSP-1 of SEQ ID NO:4. Byaligning the sequences, one skilled in the art can identifycorresponding residues, for example, using conserved and identical aminoacid residues as guides. In general, to identify correspondingpositions, the sequences of amino acids are aligned so that the highestorder match is obtained (see, e.g.: Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo et al. (1988) SIAM J AppliedMath 48:1073). Alternatively, the skilled person can number the residuesby chymotrypsin number, and thereby identify corresponding residues. Forclosely related sequences, a computer algorithm is not needed; alignmentcan be done visually.

As used herein, a “global alignment” is an alignment that aligns twosequences, from beginning to end, aligning each letter in each sequenceonly once. An alignment is produced, regardless of whether or not thereis similarity or identity between the sequences. For example, 50%sequence identity based on “global alignment” means that in an alignmentof the full sequence of two compared sequences each of 100 nucleotidesin length, 50% of the residues are the same. It is understood thatglobal alignment also can be used in determining sequence identity evenwhen the length of the aligned sequences is not the same. Thedifferences in the terminal ends of the sequences will be taken intoaccount in determining sequence identity, unless the “no penalty for endgaps” is selected. Generally, a global alignment is used on sequencesthat share significant similarity over most of their length. Exemplaryalgorithms for performing global alignment include the Needleman-Wunschalgorithm (Needleman et al. (1970) J Mol. Biol. 48: 443). Exemplaryprograms for performing global alignment are publicly available andinclude the Global Sequence Alignment Tool available at the NationalCenter for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov/),and the program available atdeepc2.psi.iastate.edu/aat/align/align.html.

As used herein, a “local alignment” is an alignment that aligns twosequences, but only aligns those portions of the sequences that sharesimilarity or identity. Hence, a local alignment determines ifsub-segments of one sequence are present in another sequence. If thereis no similarity, no alignment will be returned. Local alignmentalgorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math.2: 482 (1981)). For example, 50% sequence identity based on “localalignment” means that in an alignment of the full sequence of twocompared sequences of any length, a region of similarity or identity of100 nucleotides in length has 50% of the residues that are the same inthe region of similarity or identity.

For purposes herein, sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Default parameters for the GAP program can include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) and the weighted comparison matrix of Gribskovet al. (1986) Nucl. Acids Res. 14: 6745, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Whether any two nucleic acid moleculeshave nucleotide sequences or any two polypeptides have amino acidsequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical,” or other similar variations reciting a percent identity,can be determined using known computer algorithms based on local orglobal alignment (see e.g.,wikipedia.org/wiki/Sequence_alignment_software, providing links todozens of known and publicly available alignment databases andprograms). Generally, for purposes herein sequence identity isdetermined using computer algorithms based on global alignment, such asthe Needleman-Wunsch Global Sequence Alignment tool available fromNCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PageTYPE=BlastHome); LAlign (William Pearson implementing the Huang andMiller algorithm (Adv. Appl. Math. (1991) 12:337-357)); and the programfrom Xiaoqui Huang available atdeepc2.psi.iastate.edu/aat/align/align.html. Generally, when comparingnucleotide sequences herein, an alignment with penalty for end gaps isused. Local alignment also can be used when the sequences being comparedare substantially the same length.

Therefore, as used herein, the term “identity” represents a comparisonor alignment between a test and a reference polypeptide orpolynucleotide. In one non-limiting example, “at least 90% identical to”refers to percent identities from 90% to 100% relative to the referencepolypeptide or polynucleotide. Identity at a level of 90% or more isindicative of the fact that, assuming for exemplification purposes atest and reference polypeptide or polynucleotide length of 100 aminoacids or nucleotides are compared, no more than 10% (i.e., 10 out of100) of amino acids or nucleotides in the test polypeptide orpolynucleotide differs from that of the reference polypeptides. Similarcomparisons can be made between a test and reference polynucleotides.Such differences can be represented as point mutations randomlydistributed over the entire length of an amino acid sequence or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g., 10/100 amino acid difference (approximately 90%identity). Differences also can be due to deletions or truncations ofamino acid residues. Differences are defined as nucleic acid or aminoacid substitutions, insertions or deletions. Depending on the length ofthe compared sequences, at the level of homologies or identities aboveabout 85-90%, the result can be independent of the program and gapparameters set; such high levels of identity can be assessed readily,often without relying on software.

As used herein, a disulfide bond (also called an S—S bond or a disulfidebridge) is a single covalent bond derived from the coupling of thiolgroups. Disulfide bonds in proteins are formed between the thiol groupsof cysteine residues, and stabilize interactions between polypeptidedomains.

As used herein, “coupled” or “conjugated” means attached via a covalentor noncovalent interaction.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated thatcertain nucleic acid molecules can serve as a “probe” and as a “primer.”A primer, however, has a 3′ hydroxyl group for extension. A primer canbe used in a variety of methods, including, for example, polymerasechain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR,multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer” refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable buffer, temperature and pH.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the 5′ end of a sequence to beamplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizeswith the complement of the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, the wild-type form of a polypeptide or nucleic acidmolecule is a form encoded by a gene or by a coding sequence encoded bythe gene. Typically, a wild-type form of a gene, or molecule encodedthereby, does not contain mutations or other modifications that alterfunction or structure. The term wild-type also encompasses forms withallelic variation as occurs among and between species.

As used herein, a predominant form of a polypeptide or nucleic acidmolecule refers to a form of the molecule that is the major formproduced from a gene. A “predominant form” varies from source to source.For example, different cells or tissue types can produce different formsof polypeptides, for example, by alternative splicing and/or byalternative protein processing. In each cell or tissue type, a differentpolypeptide can be a “predominant form.”

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wild type form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species, have atleast 80%, 90% or greater amino acid identity with a wild type and/orpredominant form from the same species; the degree of identity dependsupon the gene and whether comparison is interspecies or intraspecies.Generally, intraspecies allelic variants have at least or at least about80%, 85%, 90% or 95% identity or greater with a wild type and/orpredominant form, including at least or at least about 96%, 97%, 98%,99% or greater identity with a wild type and/or predominant form of apolypeptide.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude substitutions, deletions and insertions of nucleotides. Anallele of a gene also can be a form of a gene containing a mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human. Generally, species variants have about or at least 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or moresequence identity. Corresponding residues between and among speciesvariants can be determined by comparing and aligning sequences tomaximize the number of matching nucleotides or residues, for example,such that identity between the sequences is equal to or greater than95%, equal to or greater than 96%, equal to or greater than 97%, equalto or greater than 98%, or equal to or greater than 99%. The position ofinterest is then given the number assigned in the reference nucleic acidmolecule. Alignment can be effected manually or by eye, particularly,where sequence identity is greater than 80%.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively.

For purposes herein, amino acid substitutions, deletions and/orinsertions, can be made in any MTSP-1 polypeptide or catalyticallyactive fragment thereof provided that the resulting protein exhibitsprotease activity or other activity (or, if desired, such changes can bemade to eliminate activity). Modifications can be made by makingconservative amino acid substitutions and also non-conservative aminoacid substitutions. For example, amino acid substitutions that desirablyor advantageously alter properties of the proteins can be made. In oneembodiment, mutations that prevent degradation of the polypeptide can bemade. Many proteases cleave after basic residues, such as R and K; toeliminate such cleavage, the basic residue is replaced with a non-basicresidue. Interaction of the protease with an inhibitor can be blockedwhile retaining catalytic activity by effecting a non-conservativechange at the site of interaction of the inhibitor with the protease.Other activities also can be altered. For example, receptor binding canbe altered without altering catalytic activity.

Amino acid substitutions contemplated include conservativesubstitutions, such as those set forth in Table 3, which do noteliminate proteolytic activity. As described herein, substitutions thatalter properties of the proteins, such as removal of cleavage sites andother such sites also are contemplated; such substitutions are generallynon-conservative, but can be readily effected by those of skill in theart.

As used herein, suitable conservative substitutions of amino acids areknown to those of skill in this art and can be made generally withoutaltering the biological activity of the resulting molecule. Those ofskill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. Co., p. 224). Such substitutions can be made in accordance withthose set forth in Table 3 as follows:

TABLE 3 Original residue Exemplary conservative substitution Ala (A)Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; ValLys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu

Other substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell of tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

The term substantially free of cellular material includes preparationsof proteins in which the protein is separated from cellular componentsof the cells from which it is isolated or recombinantly-produced. In oneembodiment, the term substantially free of cellular material includespreparations of protease proteins having less that about 30% (by dryweight) of non-protease proteins (also referred to herein as acontaminating protein), generally less than about 20% of non-proteaseproteins or 10% of non-protease proteins or less that about 5% ofnon-protease proteins. When the protease protein or active portionthereof is recombinantly produced, it also is substantially free ofculture medium, i.e., culture medium represents less than, about, orequal to 20%, 10% or 5% of the volume of the protease proteinpreparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of protease proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of protease proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-proteasechemicals or components.

As used herein, production by recombinant means by using recombinant DNAmethods refers to the use of the well-known methods of molecular biologyfor expressing proteins encoded by cloned DNA.

As used herein, “expression” refers to the process by which polypeptidesare produced by transcription and translation of polynucleotides. Thelevel of expression of a polypeptide can be assessed using any methodknown in art, including, for example, methods of determining the amountof the polypeptide produced from the host cell. Such methods caninclude, but are not limited to, quantitation of the polypeptide in thecell lysate by ELISA, Coomassie Blue staining following gelelectrophoresis, Lowry protein assay and Bradford protein assay.

As used herein, a “host cell” is a cell that is used to receive,maintain, reproduce and/or amplify a vector. Host cells also can be usedto express the polypeptide encoded by the vector. The nucleic acidcontained in the vector is replicated when the host cell divides,thereby amplifying the nucleic acids.

As used herein, a “vector” or “plasmid” is a replicable nucleic acidfrom which one or more heterologous proteins can be expressed when thevector is transformed into an appropriate host cell. Reference to avector includes discrete elements that are used to introduceheterologous nucleic acid into cells for either expression orreplication thereof. Reference to a vector also includes those vectorsinto which a nucleic acid encoding a polypeptide or fragment thereof canbe introduced, typically by restriction digest and ligation. Referenceto a vector also includes those vectors that contain nucleic acidencoding a protease, such as a modified MTSP-1. The vector is used tointroduce the nucleic acid encoding the polypeptide into the host cellfor amplification of the nucleic acid or for expression/display of thepolypeptide encoded by the nucleic acid. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well-known to those of skill in the art. A vector also includes“virus vectors” and “viral vectors.”

As used herein, an “expression vector” includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, vector also includes “virus vectors” or “viral vectors.”Viral vectors are engineered viruses, eukaryotic and prokaryotic, thatcan contain heterologous nucleic acid, to effect transfer and expressionof thereof in host cells. Viral vectors are characterized as eukaryoticand prokaryotic based upon the host infected by the virus from which thevector is derived, and the type of RNA polymerase (eukaryotic orprokaryotic) that recognizes the viral promoters. Hence, for example, avector derived from adenovirus is a eukaryotic vector.

As used herein, an adenovirus refers to any of a group of DNA-containingviruses that cause conjunctivitis and upper respiratory tract infectionsin humans. As used herein, naked DNA refers to histone-free DNA that canbe used for vaccines and gene therapy. Naked DNA is the genetic materialthat is passed from cell to cell during a gene transfer process calledtransformation. In transformation, purified or naked DNA is taken up bythe recipient cell which will give the recipient cell a newcharacteristic or phenotype.

As used herein, “operably linked” with reference to nucleic acidsequences, regions, elements or domains means that the nucleic acidregions are functionally related to each other. For example, nucleicacid encoding a leader peptide can be operably linked to nucleic acidencoding a polypeptide, whereby the nucleic acids can be transcribed andtranslated to express a functional fusion protein, wherein the leaderpeptide effects secretion of the fusion polypeptide. In some instances,the nucleic acid encoding a first polypeptide (e.g., a leader peptide)is operably linked to nucleic acid encoding a second polypeptide and thenucleic acids are transcribed as a single mRNA transcript, buttranslation of the mRNA transcript can result in one of two polypeptidesbeing expressed. For example, an amber stop codon can be located betweenthe nucleic acid encoding the first polypeptide and the nucleic acidencoding the second polypeptide, such that, when introduced into apartial amber suppressor cell, the resulting single mRNA transcript canbe translated to produce either a fusion protein containing the firstand second polypeptides, or can be translated to produce only the firstpolypeptide. In another example, a promoter can be operably linked tonucleic acid encoding a polypeptide, whereby the promoter regulates ormediates the transcription of the nucleic acid.

As used herein, “primary sequence” refers to the sequence of amino acidresidues in a polypeptide or the sequence of nucleotides in a nucleicacid molecule.

As used herein, protein binding sequence refers to a protein or peptidesequence that is capable of specific binding to other protein or peptidesequences generally, to a set of protein or peptide sequences or to aparticular protein or peptide sequence.

As used herein, a “tag” or an “epitope tag” refers to a sequence ofamino acids, typically added to the N- or C-terminus of a polypeptide,such as a MTSP-1 provided herein. The inclusion of tags fused to apolypeptide can facilitate polypeptide purification and/or detection.Typically, a tag or tag polypeptide refers to a polypeptide that hasenough residues to provide an epitope recognized by an antibody or canserve for detection or purification, yet is short enough such that itdoes not interfere with activity of the polypeptide to which it islinked. The tag polypeptide typically is sufficiently unique so that anantibody that specifically binds thereto does not substantiallycross-react with epitopes in the polypeptide to which it is linked.Epitope tagged proteins can be affinity purified using highly specificantibodies raised against the tags.

Suitable tag polypeptides generally have at least 5 or 6 amino acidresidues and usually between about 8-50 amino acid residues, typicallybetween 9-30 residues. The tags can be linked to one or more proteinsand permit detection of the protein or its recovery from a sample ormixture. Such tags are well-known and can be readily synthesized anddesigned. Exemplary tag polypeptides include those used for affinitypurification and include, Small Ubiquitin-like Modifier (SUMO) tags,FLAG tags, His tags, the influenza hemagglutinin (HA) tag polypeptideand its antibody 12CA5, (Field et al. (1988) Mol. Cell. Biol.8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, e.g., Evan et al. (1985) Molecular and CellularBiology 5:3610-3616); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al. (1990) Protein Engineering3:547-553). An antibody used to detect an epitope-tagged antibody istypically referred to herein as a secondary antibody.

As used herein, metal binding sequence refers to a protein or peptidesequence that is capable of specific binding to metal ions generally, toa set of metal ions or to a particular metal ion.

As used herein the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protease, or a domain thereof, present inthe sample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment can bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but can for example be aderivative thereof or some further substance. For example, detection ofa cleavage product of a complement protein, such as by SDS-PAGE andprotein staining with Coomassie blue.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein a biological activity of aprotease is its catalytic activity in which a polypeptide is hydrolyzed.

As used herein, equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions (such as, but not limited to, conservative changessuch as those set forth in Table 3, above) that do not substantiallyalter the activity or function of the protein or peptide. Whenequivalent refers to a property, the property does not need to bepresent to the same extent (e.g., two peptides can exhibit differentrates of the same type of enzymatic activity), but the activities areusually substantially the same. Complementary, when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, typically with less than 25%, 15% or 5%mismatches between opposed nucleotides. If necessary, the percentage ofcomplementarity will be specified. Typically the two molecules areselected such that they will hybridize under conditions of highstringency.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner, up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, a “chimeric protein” or “fusion protein” protease refersto a polypeptide operatively-linked to a different polypeptide. Achimeric or fusion protein provided herein can include one or moreproteases or a portion thereof, such as single chain protease domainsthereof, and one or more other polypeptides from any one or more oftranscriptional/translational control signals, signal sequences, a tagfor localization, a tag for purification, part of a domain of animmunoglobulin G, and/or a targeting agent. These chimeric or fusionproteins include those produced by recombinant means as fusion proteins,those produced by chemical means, such as by chemical coupling, through,for example, coupling to sulfhydryl groups, and those produced by anyother method whereby at least one protease, or a portion thereof, islinked, directly or indirectly via linker(s) to another polypeptide.

As used herein, operatively-linked when referring to a fusion proteinrefers to a protease polypeptide and a non-protease polypeptide that arefused in-frame to one another. The non-protease polypeptide can be fusedto the N-terminus or C-terminus of the protease polypeptide.

As used herein, a targeting agent is any moiety, such as a protein oreffective portion thereof, that provides specific binding of theconjugate to a cell surface receptor, which in some instances caninternalize bound conjugates or portions thereof. A targeting agent alsocan be one that promotes or facilitates, for example, affinity isolationor purification of the conjugate; attachment of the conjugate to asurface; or detection of the conjugate or complexes containing theconjugate.

As used herein, “linker” refers to short sequences of amino acids thatjoin two polypeptides (or nucleic acid encoding such polypeptides).“Peptide linker” refers to the short sequence of amino acids joining thetwo polypeptide sequences. Exemplary of polypeptide linkers are linkersjoining two antibody chains in a synthetic antibody fragment such as anscFv fragment. Linkers are well-known and any known linkers can be usedin the provided methods. Exemplary of polypeptide linkers are (Gly-Ser)namino acid sequences, with some Glu or Lys residues dispersed throughoutto increase solubility. Other exemplary linkers are described herein;any of these and other known linkers can be used with the providedcompositions and methods.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from a cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions,conditions related to environmental exposures and human behaviors, andconditions characterized by identifiable symptoms. Diseases or disordersinclude clinically diagnosed diseases as well as disruptions in thenormal state of the organism that have not been diagnosed as clinicaldisease. Diseases and disorders of interest herein are those involvingcomplement activation, including those mediated by complement activationand those in which complement activation plays a role in the etiology orpathology. Diseases and disorders of interest herein include thosecharacterized by complement activation (e.g., age-related maculardegeneration and renal delayed graft function).

As used herein, macular degeneration occurs when the small centralportion of the retina, known as the macula, deteriorates. There are twotypes of AMD: dry (atrophic) and wet (neovascular or exudative). MostAMD starts as the dry type and in 10-20% of individuals, it progressesto the wet type. Age-related macular degeneration is always bilateral(i.e., occurs in both eyes), but does not necessarily progress at thesame pace in both eyes.

As used herein, age-related macular degeneration (AMD) is aninflammatory disease that causes visual impairment and blindness inolder people. The proteins of the complement system are central to thedevelopment of this disease. Local and systemic inflammation in AMD aremediated by the deregulated action of the alternative pathway of thecomplement system.

As used herein, delayed graft function (DGF) is a manifestation of acutekidney injury (AKI) with attributes unique to the transplant process. Itoccurs post-transplant surgery. Delayed graft function (DGF) is a commoncomplication frequently defined as the need for dialysis during thefirst post-transplant week. Intrinsic renal synthesis of the thirdcomplement component C3 (C3) contributes to acute rejection by priming aT-cell-mediated response. For example, in brain dead donors, local renalC3 levels are higher at procurement and inversely related to renalfunction 14 days after transplant.

As used herein, a complement-mediated disease or disorder is anydisorder in which any one or more of the complement proteins plays arole in the disease, either due to an absence or presence of acomplement protein or complement-related protein or activation orinactivation of a complement or complement-related protein. In someembodiments, a complement-mediated disorder is one that is due to adeficiency in a complement protein(s). In other embodiments as describedherein a complement-mediated disorder is one that is due to activationor over-activation of a complement protein(s). A complement-mediateddisorder also is one that is due to the presence of any one or more ofthe complement proteins and/or the continued activation of thecomplement pathway. As used herein, “macular degeneration-relateddisorder” refers to any of a number of conditions in which the retinalmacula degenerates or becomes dysfunctional (e.g., as a consequence ofdecreased growth of cells of the macula, increased death orrearrangement of the cells of the macula (e.g., RPE cells), loss ofnormal biological function, or a combination of these events). Maculardegeneration results in the loss of integrity of the histoarchitectureof the cells and/or extracellular matrix of the normal macula and/or theloss of function of the cells of the macula. Examples of maculardegeneration-related disorder include age-related macular degeneration(AMD), geographic atrophy (GA), North Carolina macular dystrophy,Sorsby's fundus dystrophy, Stargardt's disease, pattern dystrophy, Bestdisease, dominant drusen, and malattia leventinese (radial drusen).Macular degeneration-related disorder also encompasses extramacularchanges that occur prior to, or following dysfunction and/ordegeneration of the macula. Thus, the term “macular degeneration-relateddisorder” also broadly includes any condition which alters or damagesthe integrity or function of the macula (e.g., damage to the RPE orBruch's membrane). For example, the term encompasses retinal detachment,chorioretinal degenerations, retinal degenerations, photoreceptordegenerations, RPE degenerations, mucopolysaccharidoses, rod-conedystrophies, cone-rod dystrophies and cone degenerations.

A macular degeneration-related disorder described herein includes AMD,such as, for example, a macular degeneration-related disorder treated byanti-VEGF treatment, such as, for example, anti-VEGF antibodies, orlaser treatment, or an implantable telescope.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease. Treatment also encompasses any pharmaceuticaluse of a modified MTSP-1 polypeptide and compositions provided herein.

As used herein, “prevention” or prophylaxis refers to methods in whichthe risk or probability of developing a disease or condition is reduced.

As used herein, a “therapeutic agent,” therapeutic regimen,radioprotectant, or chemotherapeutic mean conventional drugs and drugtherapies, including vaccines, which are known to those skilled in theart. Radiotherapeutic agents are well known in the art.

As used herein, “treatment” means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecompositions herein.

As used herein, “amelioration of the symptoms” of a particular diseaseor disorder by a treatment, such as by administration of apharmaceutical composition or other therapeutic, refers to anylessening, whether permanent or temporary, lasting or transient, of thesymptoms that can be attributed to or associated with administration ofthe composition or therapeutic.

As used herein, a “pharmaceutically effective agent” includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, anesthetics, vasoconstrictors, dispersing agents, andconventional therapeutic drugs, including small molecule drugs andtherapeutic proteins.

As used herein an “effective amount” of a compound or composition fortreating a particular disease is an amount that is sufficient toameliorate, or in some manner reduce the symptoms associated with thedisease. Such amount can be administered as a single dosage or can beadministered according to a regimen, whereby it is effective. The amountcan cure the disease but, typically, is administered in order toameliorate the symptoms of the disease. Typically, repeatedadministration is required to achieve a desired amelioration ofsymptoms.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect followingadministration to a subject. Hence, it is the quantity necessary forpreventing, curing, ameliorating, arresting or partially arresting asymptom of a disease or disorder.

As used herein, a “therapeutic effect” means an effect resulting fromtreatment of a subject that alters, typically improves or ameliorates,the symptoms of a disease or condition or that cures a disease orcondition.

As used herein, a “prophylactically effective amount” or a“prophylactically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that whenadministered to a subject, have the intended prophylactic effect, e.g.,preventing or delaying the onset, or reoccurrence, of disease orsymptoms, reducing the likelihood of the onset, or reoccurrence, ofdisease or symptoms, or reducing the incidence of viral infection. Thefull prophylactic effect does not necessarily occur by administration ofone dose, and can occur only after administration of a series of doses.Thus, a prophylactically effective amount can be administered in one ormore administrations.

As used herein, “administration of a non-complement protease,” such as amodified MTSP-1 protease, refers to any method in which thenon-complement protease is contacted with its substrate. Administrationcan be effected in vivo or ex vivo or in vitro. For example, for ex vivoadministration a body fluid, such as blood, is removed from a subjectand contacted outside the body with the modified non-complementprotease, such as a modified MTSP-1 protease. For in vivoadministration, the modified non-complement protease, such as a modifiedMTSP-1 protease, can be introduced into the body, such as by local,topical, systemic and/or other route of introduction. In vitroadministration encompasses methods, such as cell culture methods.

As used herein, “unit dose form” refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, “patient” or “subject” to be treated includes humans andhuman or non-human animals. Mammals include; primates, such as humans,chimpanzees, gorillas and monkeys; domesticated animals, such as dogs,horses, cats, pigs, goats and cows; and rodents such as mice, rats,hamsters and gerbils.

As used herein, a “combination” refers to any association between oramong two or more items. The association can be spatial or refer to theuse of the two or more items for a common purpose. The combination canbe two or more separate items, such as two compositions or twocollections, a mixture thereof, such as a single mixture of the two ormore items, or any variation thereof. The elements of a combination aregenerally functionally associated or related.

As used herein, a “composition” refers to any mixture of two or moreproducts or compounds (e.g., agents, modulators, regulators, etc.). Itcan be a solution, a suspension, liquid, powder, a paste, aqueous ornon-aqueous formulations or any combination thereof.

As used herein, a stabilizing agent refers to compound added to theformulation to protect either the antibody or conjugate, such as underthe conditions (e.g., temperature) at which the formulations herein arestored or used. Thus, included are agents that prevent proteins fromdegradation from other components in the compositions. Exemplary of suchagents are amino acids, amino acid derivatives, amines, sugars, polyols,salts and buffers, surfactants, inhibitors or substrates and otheragents as described herein.

As used herein, “fluid” refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass a therapeutic agent with a modified MTSP-1 polypeptide ornucleic acid molecule contained in the same or separate articles ofpackaging.

As used herein, a “kit” refers to a packaged combination, optionallyincluding reagents and other products and/or components for practicingmethods using the elements of the combination. For example, kitscontaining a modified protease polypeptide, such as a modified MTSP-1protease provided herein, or nucleic acid molecule provided herein andanother item for a purpose including, but not limited to,administration, diagnosis, and assessment of a biological activity orproperty are provided. Kits optionally include instructions for use.

As used herein, a “cellular extract” refers to a preparation or fractionwhich is made from a lysed or disrupted cell.

As used herein, “animal” includes any animal, such as, but not limitedto; primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer and sheep. Non-human animals exclude humans as the contemplatedanimal. The proteases provided herein are from any source, animal,plant, prokaryotic and fungal. Most proteases are of animal origin,including mammalian origin.

As used herein, a “single dosage” formulation refers to a formulationcontaining a single dose of therapeutic agent for direct administration.Single dosage formulations generally do not contain any preservatives.

As used herein, a multi-dose formulation refers to a formulation thatcontains multiple doses of a therapeutic agent and that can be directlyadministered to provide several single doses of the therapeutic agent.The doses can be administered over the course of minutes, hours, weeks,days or months. Multi-dose formulations can allow dose adjustment,dose-pooling and/or dose-splitting. Because multi-dose formulations areused over time, they generally contain one or more preservatives toprevent microbial growth.

As used herein, a “control” or “standard” refers to a sample that issubstantially identical to the test sample, except that it is nottreated with a test parameter, or, if it is a plasma sample, it can befrom a normal volunteer not affected with the condition of interest. Acontrol also can be an internal control. For example, a control can be asample, such as a virus, that has a known property or activity.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an” agent includes one or more agents.

As used herein, the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or if the alternatives aremutually exclusive.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. MTSP-1 STRUCTURE AND FUNCTION

MTSP-1 (also called matriptase, TADG-15, suppressor of tumorigenicity14, ST14; see SEQ ID NOs: 1, 2 and GenBank Accession NOs: AF118224 andAAD42765; U.S. Pat. No. 5,792,616; see, also Takeuchi (1999) Proc. Natl.Acad. Sci. U.S.A. 96:11054-1161) is a serine protease that cleavesproteins containing the amino acid sequenceP4(Arg/Lys)-P3(X)-P2(Ser)-P1(Arg)-P1′(Ala) andP4(X)-P3(Arg/Lys)-P2(Ser)-P1(Arg)-P1′(Ala) where X corresponds tonon-basic amino acids (Takeuchi (2000) J Biol Chem 275(34):26333-42) andcan cleave various synthetic substrates with Arginine or Lysine residuesat their P1 sites. MTSP-1 has at least three known physiologicalsubstrates, including urokinase-type plasminogen activator (u-PA),hepatocyte growth factor (HGF)/scatter factor and protease activatedreceptor-2 (PAR-2) (Takeuchi (2000) J Biol Chem 275(34):26333-42; Lee etal. (2000) J Biol Chem 275:36720-36725). MTSP-1 is found in epithelialcells, and in many cancer tissues. MTSP-1 is involved in normalembryonic development. MTSP-1 co-localizes with E-adherin, a tightjunction molecule that is necessary for normal embryonic development inmice.

Provided herein are modified membrane-type serine protease 1 (MTSP-1)polypeptides that are modified so that they cleave inhibitory sequencesin C3, such that activation of C3 into C3a and C3b fragments isinhibited. The activity/specificity of the modified MTSP-1 polypeptidesprovided herein is such that they cleave C3 with greater activity and/orspecificity or k_(cat)/K_(m) compared to the unmodified MTSP-1polypeptide, particularly of any of SEQ ID NOs: 1-4. The modified MTSP-1polypeptides also can have reduced activity or specificity or both for aphysiological substrate of the unmodified MTSP-1 polypeptide, such as,for example, proteinase-activated receptor-2 (PAR-2), urokinase-typeplasminogen activator (uPA), and/or hepatocyte growth factor (HGF).Thus, the modified MTSP-1 polypeptides provided herein inhibitcomplement activation in a complement pathway. The modified MTSP-1polypeptides also exhibit increased selectivity for cleaving C3 comparedto other MTSP-1 substrates, such as, for example, proteinase-activatedreceptor-2 (PAR-2), urokinase-type plasminogen activator (uPA), and/orhepatocyte growth factor (HGF). Therefore, the modified MTSP-1polypeptides provided herein do not exhibit undesired cleavageactivities against physiological native MTSP-1 substrates so that theydo not exhibit undesirable side effects.

1. Serine Proteases

Serine proteases (SPs), which include secreted enzymes and enzymessequestered in cytoplasmic storage organelles, have a variety ofphysiological roles, including in blood coagulation, wound healing,digestion, immune responses and tumor invasion and metastasis. Forexample, chymotrypsin, trypsin, and elastase function in the digestivetract; Factor 10, Factor 11, Thrombin, and Plasmin are involved inclotting and wound healing; and C1r, C1s, and the C3 convertases play arole in complement activation.

A class of cell surface proteins designated type II transmembrane serineproteases are proteases which are membrane-anchored proteins withextracellular domains. As cell surface proteins, they play a role inintracellular signal transduction and in mediating cell surfaceproteolytic events. Other serine proteases are membrane bound andfunction in a similar manner. Others are secreted. Many serine proteasesexert their activity upon binding to cell surface receptors, and, henceact at cell surfaces. Cell surface proteolysis is a mechanism for thegeneration of biologically active proteins that mediate a variety ofcellular functions.

Serine proteases, including secreted and transmembrane serine proteases,are involved in processes that include neoplastic development andprogression. While the precise role of these proteases has not beenfully elaborated, serine proteases and inhibitors thereof are involvedin the control of many intra- and extracellular physiological processes,including degradative actions in cancer cell invasion and metastaticspread, and neovascularization of tumors that are involved in tumorprogression. Proteases are involved in the degradation and remodeling ofextracellular matrix (ECM) and contribute to tissue remodeling, and arenecessary for cancer invasion and metastasis. The activity and/orexpression of some proteases have been shown to correlate with tumorprogression and development.

More than 20 families (denoted S1-S27) of serine protease have beenidentified, and they are grouped into 6 clans (SA, SB, SC, SE, SF andSG) on the basis of structural similarity and other functional evidence(Rawlings N D et al. (1994) Meth. Enzymol. 244: 19-61). There aresimilarities in the reaction mechanisms of several serine peptidases.Chymotrypsin, subtilisin and carboxypeptidase C clans have a catalytictriad of serine, aspartate and histidine in common: serine acts as anucleophile, aspartate as an electrophile, and histidine as a base. Thegeometric orientations of the catalytic residues are similar betweenfamilies, despite different protein folds. The linear arrangements ofthe catalytic residues commonly reflect clan relationships. For examplethe catalytic triad in the chymotrypsin clan (SA) is ordered HDS, but isordered DHS in the subtilisin clan (SB) and SDH in the carboxypeptidaseclan (SC).

Examples of serine proteases of the chymotrypsin superfamily includetissue-type plasminogen activator (tPA), trypsin, trypsin-like protease,chymotrypsin, plasmin, elastase, urokinase (or urinary-type plasminogenactivator, u-PA), acrosin, activated protein C, C1 esterase, cathepsinG, chymase, and proteases of the blood coagulation cascade includingkallikrein, thrombin, and Factors VIIa, IXa, Xa, XIa, and XIIa (Barret,A. J., In: Proteinase Inhibitors, Ed. Barrett, A. J., Et al., Elsevier,Amsterdam, Pages 3-22 (1986); Strassburger, W. et al., (1983) FEBSLett., 157:219-223; Dayhoff, M. O., Atlas of Protein Sequence andStructure, Vol 5, National Biomedical Research Foundation, SilverSpring, Md. (1972); and Rosenberg, R. D. et al. (1986) Hosp. Prac., 21:131-147).

The activity of proteases in the serine protease family is dependent ona set of amino acid residues that form their active site. One of theresidues is always a serine; hence their designation as serineproteases. For example, chymotrypsin, trypsin, and elastase share asimilar structure and their active serine residue is at the sameposition (Ser-195) in all three. Despite their similarities, they havedifferent substrate specificities; they cleave different peptide bondsduring protein digestion. For example, chymotrypsin prefers an aromaticside chain on the residue whose carbonyl carbon is part of the peptidebond to be cleaved. Trypsin prefers a positively charged Lys or Argresidue at this position. Serine proteases differ markedly in theirsubstrate recognition properties: some are highly specific (i.e., theproteases involved in blood coagulation and the immune complementsystem); some are only partially specific (i.e., the mammalian digestiveproteases trypsin and chymotrypsin); and others, like subtilisin, abacterial protease, are completely non-specific. Despite thesedifferences in specificity, the catalytic mechanism of serine proteasesis well conserved.

The mechanism of cleavage of a target protein by a serine protease isbased on nucleophilic attack of the targeted peptidic bond by a serine.Cysteine, threonine or water molecules associated with aspartate ormetals also can play this role. In many cases the nucleophilic propertyof the group is improved by the presence of a histidine, held in a“proton acceptor state” by an aspartate. Aligned side chains of serine,histidine and aspartate build the catalytic triad common to most serineproteases. For example, the active site residues of chymotrypsin, andserine proteases that are members of the same family as chymotrypsin,such as for example MTSP-1, are Asp102, His57, and Ser195.

The catalytic domains of all serine proteases of the chymotrypsinsuperfamily have sequence homology and structural homology. The sequencehomology includes the conservation of: 1) the characteristic active siteresidues (e.g., Ser195, His57, and Asp102 in the case of trypsin); 2)the oxyanion hole (e.g., Gly193, Asp194 in the case of trypsin); and 3)the cysteine residues that form disulfide bridges in the structure(Hartley, B. S., (1974) Symp. Soc. Gen. Microbiol., 24: 151-182). Thestructural homology includes 1) a common fold characterized by two Greekkey structures (Richardson, J. (1981) Adv. Prot. Chem., 34:167-339); 2)a common disposition of catalytic residues; and 3) detailed preservationof the structure within the core of the molecule (Stroud, R. M. (1974)Sci. Am., 231: 74-88).

Throughout the chymotrypsin family of serine proteases, the backboneinteraction between the substrate and enzyme is completely conserved,but the side chain interactions vary considerably. The identity of theamino acids that contain the S1-S4 pockets of the active site determinesthe substrate specificity of that particular pocket. Grafting the aminoacids of one serine protease to another of the same fold modifies thespecificity of one to the other. Typically, the amino acids of theprotease that contain the S1-S4 pockets are those that have side chainswithin 4 to 5 angstroms of the substrate. The interactions these aminoacids have with the protease substrate are generally called “firstshell” interactions because they directly contact the substrate. There,however, can be “second shell” and “third shell” interactions thatultimately position the first shell amino acids. First shell and secondshell substrate binding effects are determined primarily by loopsbetween beta-barrel domains. Because these loops are not core elementsof the protein, the integrity of the fold is maintained while loopvariants with novel substrate specificities can be selected during thecourse of evolution to fulfill necessary metabolic or regulatory nichesat the molecular level. Typically for serine proteases, the followingamino acids in the primary sequence are determinants of specificity:195, 102, 57 (the catalytic triad); 189, 190, 191, 192, and 226 (S1);57, the loop between 58 and 64, and 99 (S2); 192, 217, 218 (S3); theloop between Cys168 and Cys180, 215, and 97 to 100 (S4); and 41 and 151(S2′), based on chymotrypsin numbering, where an amino acid in an S1position affects P1 specificity, an amino acid in an S2 position affectsP2 specificity, an amino acid in the S3 position affects P3 specificity,and an amino acid in the S4 position affects P4 specificity. Position189 in a serine protease is a residue buried at the bottom of the pocketthat determines the S1 specificity. Structural determinants for MTSP-1are listed in Table 4, with protease domains for each of the designatedproteases aligned with that of the protease domain of chymotrypsin. Thenumber underneath the Cys168-Cys182 and 60's loop column headingsindicate the number of amino acids in the loop between the two aminoacids and in the loop. The yes/no designation under the Cys191-Cys220column headings indicates whether the disulfide bridge is present in theprotease. These regions are variable within the family ofchymotrypsin-like serine proteases and represent structural determinantsin themselves.

2. Structure

MTSP-1 cDNA has been cloned from various mammalian species. ExemplaryMTSP-1 precursor polypeptides include, but are not limited to, human(SEQ ID NO:1 and encoded by SEQ ID NO:5), mouse (SEQ ID NO:12), and rat(SEQ ID NO:13) MTSP-1 polypeptides. The human MTSP-1 mRNA transcript isnormally translated to form a 855 amino acid wild-type protein (SEQ IDNO:1). The nucleic acid molecule whose sequence is set forth in SEQ IDNO:5 (see, also Genbank AF118224) encodes the 855 amino acid MTSP-1 (SEQID NO: 1, GenBank AAD42765). MTSP-1 is multidomain proteinase with aC-terminal serine proteinase domain (Friedrich et al. (2002) J Biol Chem277(3):2160). A 683 amino acid variant of the protease has beenisolated, but this protein appears to be a truncated form or anectodomain form. As described in further detail below, MTSP-1 is azymogen or proenzyme that is further processed by proteolytic cleavageat a canonical activation motif to generate a two chain mature MTSP-1polypeptide.

At least five isoforms, produced by alternative splicing, of humanMTSP-1 exist. Forms of MTSP-1 with a molecular mass of approximately 95,78, 74, 45 and 25 kDA, corresponding to the full-length protein (95kDa), residues 149-855 of SEQ ID NO: 1 (78 kDa), residues 190-855 of SEQID NO: 1 (73 kDa) or 205-855 of SEQ ID NO: 1 (74 kDa), residues 190-614of SEQ ID NO: 1 (45.7 kDa), and residues 615-855 of SEQ ID NO: 1 (26kDa), respectively, have been detected (Ge et al., (2006) J Biol Chem281:7406-7412). Allelic variants and other variants of human MTSP-1 areknown. For example, a naturally occurring variant G827R is associatedwith ichthyosis with hypotrichosis syndrome, characterized by skinhyperkeratosis (Basel-Vanagaite et al., (2007) Am J Hum Genet80:467-477). In another example, a modified MTSP-1 polypeptidecontaining the amino acid modification C731S (C122S by chymotrypsinnumbering) in the sequence of amino acids set forth in SEQ ID NO:1(corresponding to the modification C117S in the sequence of amino acidsset forth in SEQ ID NO: 2) is known; the replacement of the freecysteine reduces aggregation of the encoded protein. Additional variantsinclude those containing amino acid modifications M2851, R381S, H656A,D711A, and S805A in full-length MTSP-1 set forth in SEQ ID NO:1.

MTSP-1 is highly expressed or active in prostate, breast, and colorectalcancers, and it is said to play a role in the metastasis of breast andprostate cancer. MTSP-1 also is expressed in a variety of epithelialtissues with high levels of activity and/or expression in the humangastrointestinal tract and the prostate. Other species of MTSP-1 areknown. For example, a mouse homolog of MTSP-1 has been identified and iscalled epithin. MTSP-1 contains a transmembrane domain, two CUB domains,four LDLR repeats, and a serine protease domain (or peptidase 51 domain)between amino acids 615-854 (set forth as SEQ ID NOs:2 and 7), which ishighly conserved among all members of the peptidase 51 family of serineproteases, such as for example with chymotrypsin (SEQ ID NOs:14 and 15).MTSP-1 is synthesized as an 855 amino acid zymogen, and activated to anactive double chain enzyme form by cleavage between Arg614 and Va1615.In addition, the single chain proteolytic domain alone is catalyticallyactive and functional.

MTSP-1 belongs to the peptidase S1 family of serine proteases (alsoreferred to as the chymotrypsin family), which also includeschymotrypsin and trypsin. Generally, chymotrypsin family members sharesequence and structural homology with chymotrypsin. MTSP-1 is numberedherein according to the numbering of mature chymotrypsin, with itsprotease domain aligned with that of the protease domain of chymotrypsinand its residues numbered accordingly. Based on chymotrypsin numbering,active site residues are Asp102, His57, and Ser195. The linear aminoacid sequence can be aligned with that of chymotrypsin and numberedaccording to the β sheets of chymotrypsin. Insertions and deletionsoccur in the loops between the beta sheets, but throughout thestructural family, the core sheets are conserved. The serine proteasesinteract with a substrate in a conserved beta sheet manner. Up to 6conserved hydrogen bonds can occur between the substrate and enzyme. Allserine proteases of the chymotrypsin family have a conserved region attheir N-terminus of the protease domain that is necessary for catalyticactivity (i.e., IIGG, VVGG, or IVGG, where the first amino acid in thisquartet is numbered according to the chymotrypsin numbering and giventhe designation Ile16. This numbering does not reflect the length of theprecursor sequence).

The substrate specificity of MTSP-1 in the protease domain has beenmapped using a positional scanning synthetic combinatorial library andsubstrate phage display (Takeuchi et al. (2000) J Biol Chem 275: 26333).Cleavage residues in substrates recognized by MTSP-1 contain Arg/Lys atP4 and basic residues or Gln at P3, small residues at P2, Arg or Lys atP1, and Ala at P1′. Effective substrates contain Lys-Arg-Ser-Arg in theP4 to P1 sites, respectively. Generally, the substrate specificity forMTSP-1 reveals a trend whereby if P3 is basic, then P4 tends to benon-basic; and if P4 is basic, then P3 tends to be non-basic. Knownsubstrates for MTSP-1, including, for example, proteinase-activatedreceptor-2 (PAR-2), urokinase-type plasminogen activator (uPA), andhepatocyte growth factor (HGF), conform to the cleavage sequence forMTSP-1 specific substrates.

MTSP-1 can cleave selected synthetic substrates as efficiently astrypsin, but exhibit a more restricted specificity for substrates thantrypsin. The catalytic domain of MTSP-1 has the overall structural foldof a (chymo)trypsin-like serine protease, but displays unique propertiessuch as a hydrophobic/acidic S2/S4 subsites and an exposed 60 loop.Similarly, MTSP-1 does not indiscriminately cleave peptide substrates ataccessible Lys or Arg residues, but requires recognition of additionalresidues surrounding the scissile peptide bond. This requirement for anextended primary sequence highlights the specificity of MTSP-1 for itssubstrates. For example, although MTSP-1 cleaves proteinase activatedreceptor-2 (PAR-2) (displaying a P4 to P1 target sequence ofSer-Lys-Gly-Arg), the enzyme does not activate proteins closely relatedto this substrate such as PAR-1, PAR-3, and PAR-4 that do not displaytarget sequences matching the extended MTSP-1 specificity near thescissile bond (see Friedrich et al. (2002) J Biol Chem 277: 2160).

The protease domain of MTSP-1 (see, e.g., SEQ ID NOs: 2, 4) is composedof a pro-region and a catalytic domain. The catalytically active portionof the polypeptide begins after the autoactivation site at amino acidresidue 611 of the mature protein (see, e.g., SEQ ID NOs: 1, 3 at RQARfollowed by the residues VVGG). The S1 pocket of MTSP-1 and trypsin aresimilar with good complementarity for Lys as well as Arg P1 residues,thereby accounting for some similarities in substrate cleavage withtrypsin. The accommodation of the P1-Lys residues is mediated by Ser190whose side chain provides an additional hydrogen bond acceptor tostabilize the buried α-ammonium group (see Friedrich et al. (2002) JBiol Chem 277: 2160). The S2 pocket is shaped to accommodate small tomedium-sized hydrophobic side chains of P2 amino acids and generallyaccepts a broad range of amino acids at the P2 position. Upon substratebinding, the S2 sub-site is not rigid as evidenced by the rotation ofthe Phe99 benzyl group. Association of the substrate amino acids atpositions P3 (for either Gln or basic residues) and P4 (for Arg or Lysresidues) appears to be mediated by electrostatic interactions in the S3and S4 pockets with the acidic side chains of Asp-217 and/or Asp-96,which can favorably pre-orient specific basic peptide substrates as theyapproach the enzyme active site cleft. The side chain of a P3 residuealso is able to hydrogen bond the carboxamide group of Gln192 oralternatively, the P3 side chain can extend into the S4 sub-site to forma hydrogen bond with Phe97 thereby weakening the inter-main chainhydrogen bonds with Gly216. In either conformation, a basic P3 sidechain is able to interact favorably with the negative potential of theMTSP-1 S4 pocket. The mutual charge compensation and exclusion from thesame S4 site explains the low probability of the simultaneous occurrenceof Arg/Lys residues at P3 and P4 in good MTSP-1 substrates. Generally,the amino acid positions of MTSP-1 (based on chymotrypsin numbering)that contribute to extended specificity for substrate binding include:146 and 151 (S1′); 189, 190, 191, 192, 216, 226 (S1); 57, 58, 59, 60,61, 62, 63, 64, 99 (S2); 192, 217, 218, 146 (S3); 96, 97, 98, 99, 100,168, 169, 170, 170A, 171, 172, 173, 174, 175, 176, 178, 179, 180, 215,217, 224 (S4). Table 4 summarizes the residues in MTSP-1 for some of theamino acid positions important for specificity interactions with atargeted substrate. Typically, modification of an MTSP-1 protease toalter any one or more of the amino acids in the extended specificitybinding pocket or other secondary sites of interaction affect thespecificity or selectivity of a protease for a target substrate.

TABLE 4 Structural Determinants for MTSP-1 substrate cleavage(chymotrypsin numbering) Residues that Determine Specificity S4 S3 S2 S1171 174 180 215 Cys168 192 218 99 57 60’s 189 190 226 Cys191 Cys182 loopCys20 (58- 64) Leu Gln Met Trp 13* Gln Asp Phe His 16* Asp Ser Gly yes*number of residues

3. Function/Activity

Membrane type serine protease 1 (MTSP-1) is a serine protease normallyexpressed in epithelial tissues, including the skin, and in kidney,lung, prostate and mammary epithelium (Kim et al. (1999) Immunogenetics49:420-428; Oberst et al. (2001) Am J Pathol 158:1301-1311; Takeguchi etal. (1999) Proc Natl Acad Sci 96:11054-11061) and expressed in a varietyof tumor types (Oberst et al. (2001) Am J Pathol 158:1301-1311). MTSP-1is essential for post-natal survival; MTSP-1 deficient mice developed toterm but died shortly thereafter and were characterized by aberrant skindevelopment, implicating a role for MTSP-1 in epithelial and epidermalbarrier function and as important for normal skin and hair development(List et al. (2002) Oncogene 21(23):3765-3779). Post-natal MTSP-1ablation caused loss of tight junction formation and mislocalizedtight-junction associated proteins in mutant animals (List et al. (2009)Am J Pathology 175:1453-1463), implicating MTSP-1 as essential inmaintenance of mouse epithelia. MTSP-1 also is reported to promoteneural progenitor cell migration (Kendall et al., (2008) Stem Cells26(6):1575-86).

MTSP-1 is highly expressed or active in prostate, breast, lung, ovaryand colorectal cancers and it may play a role in the metastasis ofbreast and prostate cancer. MTSP-1 also can be identified in bloodvessels associated with tumors. MTSP-1 also is expressed in a variety ofepithelial tissues with high levels of activity and/or expression in thehuman gastrointestinal tract and the prostate. MTSP-1 presence on thetumor or epithelial cell surface allows for interaction with a varietyof factors and for proteolytic digestion of a broad range of substrates.

The role of MTSP-1 in cell migration on tumor activity may inducechanges in the extracellular environment and the surrounding cellscontributing to cell migration, progression, and metastasis. Because ofthe role of MTSP-1 in vascular diseases and cancer, MTSP-1 polypeptidesprovided herein are altered such that they exhibit reduced selectivitytowards these proteins.

C. COMPLEMENT INHIBITION BY TARGETING C3

The modified MTSP-1 polypeptides provided herein exhibit increasedspecificity and/or activity for an inhibitory cleavage sequence incomplement protein C3 compared to MTSP-1 polypeptides not containing theamino acid modifications (e.g., wild type human MTSP-1 (see, SEQ IDNO:1) or a reference full-length human MTSP-1 (see, SEQ ID NO:3) or thecatalytic domain or protease domain thereof (see, SEQ ID NO:2 or 4)).The reference MTSP-1 polypeptides include the replacement C122S, bychymotrypsin numbering. Replacement with S at residue 122 does not alterspecificity or activity on C3, but reduces aggregation. Since C3 isinvolved in the 3 initiation pathways of complement (see, e.g., FIG. 1), targeting C3 by proteolytic inhibition provides a general and broadtherapeutic target for inactivation of the complement cascade.Inactivation cleavage of C3 blocks terminal activity of complement aswell as the alternative pathway amplification loop. All three pathwaysconverge at C3 (see, e.g., FIG. 1 ). By virtue of the ability to inhibitcomplement activation, such modified MTSP-1 polypeptides can be used totreat various diseases, conditions and pathologies associated withcomplement activation, such as inflammatory responses and autoimmunediseases. Complement activation is associated with the development ofdiseases and conditions by promoting local inflammation and damage totissues caused in part by the generation of effector molecules and amembrane attack complex. In one example, such as in many autoimmunediseases, complement produces tissue damage because it is activatedunder inappropriate circumstances such as by antibody to host tissues.In other situations, complement can be activated normally, such as bysepticemia, but still contributes to disease progression, such as inrespiratory distress syndrome. Pathologically, complement can causesubstantial damage to blood vessels (vasculitis), kidney basementmembrane and attached endothelial and epithelial cells (nephritis),joint synovium (arthritis), and erythrocytes (hemolysis) if notadequately controlled. The role of C3 in complement activation isdiscussed in further detail below.

The modified MTSP-1 polypeptides herein can cleave C3. For example, Asingle intravenous injection of anti-C3 MTSP-1 variants can eliminate C3from plasma in vivo; and similarly, a single intravitreal injection cancleave all C3 present in vitreous humor. Since C3 is the first componentof the common complement pathway that is required for complementactivation via all three “initiation” pathways (classical, alterative,and lectin), inactivation/elimination of C3 is functionally relevant forall three complement pathways.

1. Complement Protein C3 and its Role in Initiating Complement

The complement system involves over 30 soluble and cell-membrane boundproteins that function not only in the antibody-mediated immuneresponse, but also in the innate immune response to recognize and killpathogens such as bacteria, virus-infected cells, and parasites.Complement activation is initiated on pathogen surfaces through threedistinct pathways: the classical pathway, the alternative pathway, andthe lectin pathway. These pathways are distinct in that the componentsrequired for their initiation are different, but the pathways ultimatelygenerate the same set of effector molecules (e.g., C3 convertases) whichcleave complement protein C3 to trigger the formation of the membraneattack complex (MAC) (see, e.g., FIG. 1 ). Thus, complement protein C3is an attractive target for a therapeutic since modulation of C3 resultsin modulation of various opsonins, anaphylatoxins and the MAC. Further,naturally occurring complement inhibitor proteins including factor H(FH), CR1, complement receptor Ig (CR1g), DAF and MCP inhibit at the C3level.

There are three (3) pathways of complement activation (See, FIG. 1 ,which depicts these pathways). The pathways of complement are distinct;each relies on different molecules and mechanisms for initiation. Thepathways are similar in that they converge to generate the same set ofeffector molecules, i.e., C3 convertases. In the classical and lectinpathways C4b2b acts as a C3 convertase; in the alternative pathway,C3bBb is a C3 convertase (see Table 5). Cleavage of C3 generates C3b,which acts as an opsonin and as the main effector molecule of thecomplement system for subsequent complement reactions, and C3a, which isa peptide mediator of inflammation. The addition of C3b to each C3convertase forms a C5 convertase that generates C5a and C5b. C5a, likeC3a, is a peptide mediator of inflammation. C5b mediates the “late”events of complement activation initiating the sequence of reactionsculminating in the generation of the membrane attack complex (MAC).Although the three pathways produce different C3 and C5 convertases, allof the pathways produce the split products of C3 and C5 and form MAC.Alternatively, C3 can be cleaved and activated by extrinsic proteases,such as lysosomal enzymes and elastase (Markiewski and Lambris (2007) AmJ Pathology 171:715-727; Ricklin and Lambris (2007) Nat Biotechnol25:1265-1275).

TABLE 5 Complement Cascades Alternative Classical Lectin Pathway PathwayPathway Activators Pathogen surface antigen-bound Pathogens viamolecules LPS, IgM and IgG; recognition of teichoic acid, non-immunecarbohydrates zymosan molecules on surface C3 convertase C3bBb C4b2bC4b2b C5 convertase C3bBb3b C4b2b3b C4b2b3b MAC C5678poly9 C5678poly9C5678poly9 anaphylatoxins C3a, C5a C3a, C4a, C5a C3a, C4a, C5a

a. Classical Pathway

C1q is the first component of the classical pathway of complement. C1qis a calcium-dependent binding protein associated with the collectinfamily of proteins due to an overall shared structural homology(Malhotra et al., (1994) Clin Exp Immunol. 97(2):4-9; Holmskov et al.(1994) Immunol Today 15(2):67-74). Collectins, often called patternrecognition molecules, generally function as opsonins to targetpathogens for phagocytosis by immune cells. In contrast to conventionalcollectins, such as MBL, the carboxy-terminal globular recognitiondomain of C1q does not have lectin activity but can serve as a “charged”pattern recognition molecule due to marked differences in theelectrostatic surface potential of its globular domains (Gaboriaud etal. (2003) J. Biol. Chem. 278(47):46974-46982).

C1q initiates the classical pathway of complement in two different ways.First, the classical pathway is activated by the interaction of C1q withimmune complexes (i.e. antigen-antibody complexes or aggregated IgG orIgM antibody) thus linking the antibody-mediated humoral immune responsewith complement activation. When the Fab portion (the variable region)of IgM or IgG binds antigen, the conformation of the Fc (constant)region is altered, allowing C1q to bind. C1q must bind at least 2 Fcregions to be activated. C1q, however, also is able to activatecomplement in the absence of antibody thereby functioning in the innateor immediate immune response to infection. Besides initiation by anantibody, complement activation also is achieved by the interaction ofC1q with non-immune molecules such as polyanions (bacteriallipopolysaccharides, DNA, and RNA), certain small polysaccharides, viralmembranes, C reactive protein (CRP), serum amyloid P component (SAP),and bacterial, fungal and viral membrane components.

C1q is part of the C1 complex which contains a single C1q molecule boundto two molecules each of the zymogens C1r and C1s. Binding of more thanone of the C1q globular domains to a target surface (such as aggregatedantibody or a pathogen), causes a conformational change in the(C1r:C1s)₂ complex which results in the activation of the C1r proteaseto cleave C1s to generate an active serine protease. Active C1s cleavessubsequent complement components C4 and C2 to generate C4b and C2b,which together form the C3 convertase of the classical pathway. The C3convertase cleaves C3 into C3b, which covalently attaches to thepathogen surface and acts as an opsonin, and C3a, which stimulatesinflammation. Some C3b molecules associate with C4b2b complexes yieldingC4b2b3b which is the classical cascade C5 convertase. Table 6 summarizesthe proteins involved in the classical pathway of complement.

TABLE 6 Proteins of the Classical Pathway Native Active Component FormFunction of the Active Form C1 C1q Binds directly to pathogen surfacesor (C1q:(C1r:C1s)₂) indirectly to antibody bound to pathogens C1rCleaves C1s to an active protease C1s Cleaves C4 and C2 C4 C4b Binds topathogen and acts as an opsonin; binds C2 for cleavage by C1s C4aPeptide mediator of inflammation C2 C2b Active enzyme of classicalpathway C3/C5 convertase; cleaves C3 and C5 C2a Precursor of vasoactiveC2 kinin C3 C3b Binds to pathogen surfaces and acts as an opsonin;initiates amplification via the alternative pathway; binds C5 forcleavage by C2b C3a Peptide mediator of inflammation

b. Alternative Pathway

The alternative pathway is initiated by foreign pathogens in the absenceof antibody. Initiation of complement by the alternative pathway occursthrough the spontaneous hydrolysis of C3 into C3b. A small amount of C3bis always present in body fluids, due to serum and tissue proteaseactivity. Host self-cells normally contain high levels of membranesialic acid which inactivate C3b if it binds, but bacteria contain lowexternal sialic acid levels and thereby bind C3b without inactivatingit. C3b on pathogen surfaces is recognized by the protease zymogenFactor B. Factor B is cleaved by Factor D. Factor D is the onlyactivating protease of the complement system that circulates as anactive enzyme rather than as a zymogen, but since Factor B is the onlysubstrate for Factor D the presence of low levels of an active proteasein normal serum is generally safe for the host. Cleavage of Factor B byFactor D yields the active product Bb which can associate with C3b toform C3bBb, the C3 convertase of the alternative pathway. Similar to theclassical pathway, the C3 convertase produces more C3b and C3a from C3.C3b covalently attaches to the pathogen surface and acts as an opsoninand additionally initiates the alternative pathway, while C3a stimulatesinflammation. Some C3b joins the complex to form C3bBb3b, thealternative pathway C5 convertase. C3bBb3b is stabilized by the plasmaprotein properdin or Factor P which binds to microbial surfaces andstabilizes the convertase. Table 7 summarizes the proteins involved inthe alternative pathway of complement.

TABLE 7 Proteins of the Alternative Pathway Native Active Component FormFunction of the Active Form C3 C3b Binds to pathogen surface, bindsFactor B for cleavage by Factor D Factor B Ba Small fragment of FactorB, unknown function Bb Active enzyme of the C3 convertase and C5convertase Factor D D Plasma serine protease, cleaves Factor B when itis bound to C3b to Ba and Bb Factor P P Plasma proteins with affinityfor C3bBb (properdin) convertase on bacterial cells; stabilizesconvertase

c. Lectin Pathway

The lectin pathway (also referred to as the MBL pathway) is initiatedfollowing recognition and binding of pathogen-associated molecularpatterns (PAMPs; i.e., carbohydrates moieties) by lectin proteins.Examples of lectin proteins that activate the lectin pathway ofcomplement include mannose binding lectin (MBL) and ficolins (i.e.L-ficolin, M-ficolin, and H-ficolin). MBL is a member of the collectinfamily of proteins and thereby exists as an oligomer of subunitscomposed of identical polypeptide chains each of which contains acysteine-rich, a collagen-like, a neck, and a carbohydrate-recognitionor lectin domain. MBL acts as a pattern recognition molecule torecognize carbohydrate moieties, particularly neutral sugars such asmannose or N-acetylglucosamine (GlcNAc) on the surface of pathogens viaits globular lectin domain in a calcium-dependent manner. MBL also actsas an opsonin to facilitate the phagocytosis of bacterial, viral, andfungal pathogens by phagocytic cells. Additional initiators of thelectin pathway include the ficolins including L-ficolin, M-ficolin, andH-ficolin (see e.g., Liu et al. (2005) J Immunol. 175:3150-3156).Similar to MBL, ficolins recognize carbohydrate moieties such as, forexample, N-acetyl glucosamine and mannose structures.

The activation of the alternative pathway by MBL or ficolins isanalogous to activation of the classical pathway by C1q whereby a singlelectin molecule interacts with two protease zymogens. In the case of thelectin proteins, the zymogens are MBL-associated serine proteases,MASP-1 and MASP-2, which are closely homologous to the C1r and C1szymogens of the classical pathway. Upon recognition of a PAMP by alectin protein, such as for example by binding to a pathogen surface,MASP-1 and MASP-2 are activated to cleave C4 and C2 to form the MBLcascade C3 convertase. C3b then joins the complex to form the MBLcascade C5 convertase. MASP activation is implicated not only inresponses to microorganisms, but in any response that involves exposingneutral sugars, including but not limited to tissue injury, such as thatobserved in organ transplants. Like the alternative cascade, the MBLcascade is activated independent of antibody; like the classicalcascade, the MBL cascade utilizes C4 and C2 to form C3 convertase. Table8 summarizes the proteins involved in the lectin pathway of complement.

TABLE 8 Proteins of the Lectin Pathway Native Component Active FormFunction of the Active Form MBL MBL Recognizes PAMPs, such as onpathogen surfaces (e.g., via recognition of carbohydrates) FicolinsL-Ficolin; Recognizes PAMPs, such as on pathogen M-Ficolin, or surfaces(e.g., via recognition of H-Ficolin carbohydrates) MASP-1 MASP-1 CleavesC4 and C2 MASP-2 MASP-2 Cleaves C4 and C2

d. Complement-Mediated Effector Functions

Regardless of which initiation pathway is used, the end result is theformation of activated fragments of complement proteins (e.g. C3a, C4a,and C5a anaphylatoxins and C5b-9 membrane attack complexes), which actas effector molecules to mediate diverse effector functions. Therecognition of complement effector molecules by cells for the initiationof effector functions (e.g. chemotaxis and opsonization) is mediated bya diverse group of complement receptors. The complement receptors aredistributed on a wide range of cell types including erythrocytes,macrophages, B cells, neutrophils, and mast cells. Upon binding of acomplement component to the receptor, the receptors initiate anintracellular signaling cascade resulting in cell responses such asstimulating phagocytosis of bacteria and secreting inflammatorymolecules from the cell. For example, the complement receptors CR1 andCR2 which recognize C3b, C4b, and their products are important forstimulating chemotaxis. CR3 (CD11b/CD18) and CR4 (CD11c/CD18) areintegrins that are similarly important in phagocytic responses but alsoplay a role in leukocyte adhesion and migration in response to iC3b. TheC5a and C3a receptors are G protein-coupled receptors that play a rolein many of the pro-inflammatory-mediated functions of the C5a and C3aanaphylatoxins. For example, receptors for C3a, C3aR, exist on mastcells, eosinophils, neutrophils, basophils and monocytes and aredirectly involved in the pro-inflammatory effects of C3a.

Thus, through complement receptors, these complement effector moleculefragments mediate several functions including leukocyte chemotaxis,activation of macrophages, vascular permeability and cellular lysis(Frank, M. and Fries, L. Complement. In Paul, W. (ed.) FundamentalImmunology, Raven Press, 1989). A summary of some effector functions ofcomplement products are listed in Table 9.

TABLE 9 Complement Effector Molecules and Functions Product Activity C2b(prokinin) accumulation of body fluid C3a basophil and mast celldegranulation; (anaphylatoxin) enhanced vascular permeability; smoothmuscle contraction; Induction of suppressor T cells C3b and itsopsonization; phagocyte activation products C4a basophil & mast cellactivation; smooth (anaphylatoxin) muscle contraction; enhanced vascularpermeability C4b opsonization C5a basophil & mast cell activation;enhanced (anaphylatoxin; vascular permeability; smooth musclechemotactic contraction; chemotaxis; neutrophil factor) aggregation;oxidative metabolism stimulation; stimulation of leukotriene release;induction of helper T-cells C5b67 chemotaxis; attachment to other cellmembranes and lysis of bystander cells C5b6789 lysis of target cells(C5b-9)i. Complement-Mediated Lysis: Membrane Attack Complex

The final step of the complement cascade by all three pathways is theformation of the membrane attack complex (MAC) (FIG. 1 ). C5 can becleaved by any C5 convertase into C5a and C5b. C5b combines with C6 andC7 in solution, and the C5b67 complex associates with the pathogen lipidmembrane via hydrophobic sites on C7. C8 and several molecules of C9,which also have hydrophobic sites, join to form the membrane attackcomplex, also called C5b6789 or C5b-9. C5b-9 forms a pore in themembrane through which water and solutes can pass, resulting in osmoticlysis and cell death. If complement is activated on an antigen without alipid membrane to which the C5b67 can attach, the C5b67 complex can bindto nearby cells and initiate bystander lysis. A single MAC can lyse anerythrocyte, but nucleated cells can endocytose MAC and repair thedamage unless multiple MACs are present. Gram negative bacteria, withtheir exposed outer membrane and enveloped viruses, are generallysusceptible to complement-mediated lysis. Less susceptible are Grampositive bacteria, whose plasma membrane is protected by their thickpeptidoglycan layer, bacteria with a capsule or slime layer around theircell wall, or viruses which have no lipid envelope. Likewise, the MACcan be disrupted by proteins that bind to the complex before membraneinsertion such as Streptococcal inhibitor of complement (SIC) andclusterin. Typically, the MAC helps to destroy gram-negative bacteria aswell as human cells displaying foreign antigens (virus-infected cells,tumor cells, etc.) by causing their lysis and also can damage theenvelope of enveloped viruses.

ii. Inflammation

Inflammation is a process in which blood vessels dilate and become morepermeable, thus enabling body defense cells and defense chemicals toleave the blood and enter the tissues. Complement activation results inthe formation of several proinflammatory mediators such as C3a, C4a andC5a. The intact anaphylatoxins in serum or plasma are quickly convertedinto the more stable, less active C3a-desArg, C4a-desArg, or C5a-desArgforms, by carboxypeptidase N. C3a, C4a and C5a, and to a lesser extenttheir desArg derivatives, are potent bioactive polypeptides, termedanaphylatoxins because of their inflammatory activity. Anaphylatoxinsbind to receptors on various cell types to stimulate smooth musclecontraction, increase vascular permeability, and activate mast cells torelease inflammatory mediators. C5a, the most potent anaphylatoxin,primarily acts on white blood cells, particularly neutrophils. C5astimulates leukocyte adherence to blood vessel walls at the site ofinfection by stimulating the increased expression of adhesion moleculesso that leukocytes can squeeze out of the blood vessels and into thetissues, a process termed diapedesis. C5a also stimulates neutrophils toproduce reactive oxygen species for extracellular killing, proteolyticenzymes, and leukotrienes. C5a also can further amplify the inflammatoryprocess indirectly by inducing the production of chemokines, cytokines,and other proinflammatory mediators. C5a also interacts with mast cellsto release vasodilators such as histamine so that blood vessels becomemore permeable. C3a also interacts with white blood cells, with majoreffects on eosinophils indicating a role for C3a in allergicinflammation. C3a induces smooth muscle contraction, enhances vascularpermeability, and causes degranulation of basophils and release ofhistamine and other vasoactive substances. C2a can be converted to C2kinin, which regulates blood pressure by causing blood vessels todilate.

Although technically not considered an anaphylatoxin, iC3b, an inactivederivative of C3b, functions to induce leukocyte adhesion to thevascular endothelium and induce the production of the pro-inflammatorycytokine IL-1 via binding to its cell surface integrin receptors. C5b-9also indirectly stimulates leukocyte adhesion, activation, andchemotaxis by inducing the expression of cell adhesion molecules such asE-selectin, and inducing interleukin-8 secretion (Bhole et al. (2003)Crit Care Med 31(1):97-104). C5b-9 also stimulates the release ofsecondary mediators that contribute to inflammation, such as forexample, prostaglandin E₂, leukotriene B₄, and thromboxane.

Conversion of the human complement components C3 and C5 to yield theirrespective anaphylatoxin products has been implicated in certainnaturally occurring pathologic states including: autoimmune disorderssuch as systemic lupus erythematosus, rheumatoid arthritis, malignancy,myocardial infarction, Purtscher's retinopathy, sepsis and adultrespiratory distress syndrome. In addition, increased circulating levelsof C3a and C5a have been detected in certain conditions associated withiatrogenic complement activation such as: cardiopulmonary bypasssurgery, renal dialysis, and nylon fiber leukaphoresis.

iii. Chemotaxis

Chemotaxis is a process by which cells are directed to migrate inresponse to chemicals in their environment. In the immune response, avariety of chemokines direct the movement of cells, such as phagocyticcells, to sites of infection. For example, C5a is the main chemotacticfactor for circulating neutrophils, but also can induce chemotaxis ofmonocytes. Phagocytes will move towards increasing concentrations of C5aand subsequently attach, via their CR1 receptors, to the C3b moleculesattached to the antigen. The chemotactic effect of C5a, observed withbasophils, eosinophils, neutrophils, and mononuclear phagocytes, isactive at concentrations as low as 10⁻¹⁰ M.

iv. Opsonization

An important action of complement is to facilitate the uptake anddestruction of pathogens by phagocytic cells. This occurs by a processtermed opsonization whereby complement components bound to targetbacteria interact with complement receptors on the surface of phagocyticcells such as neutrophils or macrophages. In this instance, thecomplement effector molecules are termed opsonins. Opsonization ofpathogens is a major function of C3b and C4b. iC3b also functions as anopsonin. C3a and C5a increase the expression of C3b receptors onphagocytes and increase their metabolic activity.

C3b and, to a lesser extent, C4b help to remove harmful immune complexesfrom the body. C3b and C4b attach the immune complexes to CR1 receptorson erythrocytes. The erythrocytes then deliver the complexes to fixedmacrophages within the spleen and liver for destruction. Immunecomplexes can lead to a harmful Type III hypersensitivity.

v. Activation of the Humoral Immune Response

Activation of B cells requires ligation of the B cell receptor (BCR) byantigen. It has been shown, however, that complement plays a role inlowering the threshold for B cell responses to antigen by up to1000-fold. This occurs by the binding of C3d or C3dg, complementproducts generated from the breakdown fragments of C3, to CR2 receptorson B-lymphocytes which can co-ligate with the BCR. Co-ligation occurswhen antigenic particles, such as for example immune complexes,opsonized with C3d bind the CR2 receptor via C3d as well as the BCRthrough antigen. Co-ligation of antigen complexes also can occur whenC3d binds to antigens enhancing their uptake by antigen presentingcells, such as dendritic cells, which can then present the antigen to Bcells to enhance the antibody response. Mice deficient in CR2 displaydefects in B cell function that result in reduced levels of naturalantibody and impaired humoral immune responses.

2. C3 Structure and Function

The variant MTSP-1 polypeptides provided herein cleave complementprotein C3 or its proteolytic fragments thereby inhibiting complement.Human complement protein C3 (Uniprot Accession No. P01024) is a 1663amino acid single chain pre-proprotein having an amino acid sequence setforth in SEQ ID NO:9. The protein is encoded by a 41 kb gene located onchromosome 19 (nucleotide sequence set forth in SEQ ID NO:10). Thepre-proprotein contains a 22 amino acid signal peptide (amino acids 1-22of SEQ ID NO:9) and a tetra-arginine sequence (amino acids 668-671 ofSEQ ID NO:9) that is removed by a furin-like enzyme resulting information of a mature two chain protein containing a beta chain (aminoacids 23-667 of SEQ ID NO:9) and an alpha chain (amino acids 672-1663 ofSEQ ID NO:9), that are linked by an interchain disulfide bond betweenamino acid residues Cys559 and Cys816. The mature 2 chain protein has asequence of amino acids set forth in SEQ ID NO:16.

During the complement cascade, complement protein C3 is furtherprocessed by proteolytic cleavage to form various C3 proteolyticfragments. As described above, all three complement initiation pathwaysconverge on the C3 convertases C4b2b and C3bBb. C3 convertases cleave C3between residues 748 and 749 of SEQ ID NO:9 (see Table 10 below)generating the anaphylatoxin C3a (amino acids 672-748 of SEQ ID NO: 9)and the opsonin C3b (C3b alpha′ chain; amino acids 749-1663 of SEQ IDNO: 9). C3a is involved in inflammation and C3b forms the C5 convertasesultimately leading to C5a anaphylatoxin and the MAC. The variant MTSP-1polypeptides provided herein inhibit complement, and as such, do notcleave C3 at this GLAR cleavage site.

C3b has binding sites for various complement components including C5,properdin (P), factors H, B and I, complement receptor 1 (CR1) and themembrane co-factor protein (MCP) (see Sahu and Lambris (2001)Immunological Reviews 180:35-48). Binding of factor I, a plasmaprotease, in the presence of cofactors H, CR1 and MCP results ininactivation of C3b whereas binding of factors B and P in the presenceof factor D results in amplification of C3 convertase and initiation ofMAC. Factor I cleaves C3b in the presence of cofactors between residues1303-1304, 1320-1321 and 954-955 of SEQ ID NO:9 (see Table 10 below)generating fragments iC3b (amino acids 749-1303 of SEQ ID NO: 9) and C3f(amino acids 1304-1320 of SEQ ID NO: 9). Factor I subsequently cleavesiC3b generating C3c (C3c alpha′ chain fragment 1; amino acids 749-954 ofSEQ ID NO: 9) and C3dg (amino acids 955-1303 of SEQ ID NO: 9). The endresult is that C3b is permanently inactivated (see Sahu and Lambris(2001) Immunological Reviews 180:35-48). Since Factor I inactivates C3b,the factor I cleavage sites are ideal candidates for cleavage by thevariant MTSP-1 polypeptides provided herein. Additional C3b proteolyticfragments include C3g (amino acids 955-1001 of SEQ ID NO: 9), C3d (aminoacids 1002-1303 of SEQ ID NO: 9), and C3c alpha′ chain fragment 2 (aminoacids 1321-1663 of SEQ ID NO: 9). Cleavage sequences in complementprotein C3 are set forth in Table 10 below, which lists the P4-P1residues, the amino acid residues of the cleavage site (P1-P1′ site) andthe protease responsible for cleavage. The modified MTSP-1 polypeptidesprovided herein do not cleave at these sites.

TABLE 10 Complement Protein C3 Cleavage Sequences P4-P1 Cleavage SiteResidues (in SEQ ID NO: 9) Protease SEQ ID NO GLAR 748-749 C3 convertase17 RLGR 954-955 Factor I 18 LPSR 1303-1304 Factor I 19 SLLR 1320-1321Factor I 20

a. C3a

C3a (amino acids 672-748 of SEQ ID NO:9) is an anaphylatoxin that isinvolved in inflammation, basophil and mast cell degranulation, enhancedvascular permeability, smooth muscle contraction and induction ofsuppressor T cells.

b. C3b

C3b (amino acids 749-1663 of SEQ ID NO:9) has various roles in thecomplement cascade. C3b is an opsonin that facilitates the uptake anddestruction of pathogens by phagocytic cells. Additionally, C3b combineswith the C3 convertases to generate the C5 convertases which activatecomplement protein C5 thereby generating the C5a anaphylatoxin and C5b,which combines with C6, C7, C8 and C9 to form the membrane attackcomplex. Furthermore, as described in section 1b above, C3b is involvedin the alternative pathway of complement initiation. C3b is regulated bycomplement regulatory protein Factor I, a plasma protease which degradesC3b into various fragments, including iC3b, C3c, C3d, C3f and C3dg,thereby permanently inactivating C3b.

C3b plays a critical role in complement-mediated effector functions byvirtue of its ability to bind to the C3 convertases C4b2b and C3bBbthereby generating the C5 convertases C4b2b3b and C3bBb3b. The C5convertases cleave the zymogen C5 into its active fragments, namely theC5a anaphylatoxin and C5b. C5a is involved in chemotaxis andinflammation and C5b is involved in formation of MAC.

i. Inhibitors of C3b

C3b has binding sites for various complement components including C5,properdin (P), factors H, B and I, complement receptor 1 (CR1) and themembrane co-factor protein (MCP) (see Sahu and Lambris (2001)Immunological Reviews 180:35-48). Binding of factor I, a plasmaprotease, in the presence of cofactors H, CR1 and MCP results ininactivation of C3b whereas binding of factors B and P in the presenceof factor D results in amplification of C3 convertase and initiation ofMAC. Factor I cleaves C3b in the presence of cofactors between residues1303-1304, 1320-1321 and 954-955 of SEQ ID NO:9 generating fragmentsiC3b (amino acids 749-1303 of SEQ ID NO:9) and C3f (amino acids1304-1320 of SEQ ID NO:9). Although technically not considered ananaphylatoxin, iC3b, an inactive derivative of C3b, functions to induceleukocyte adhesion to the vascular endothelium and induce the productionof the pro-inflammatory cytokine IL-1 via binding to its cell surfaceintegrin receptors. In addition, iC3b functions as an opsonin. Factor Isubsequently cleaves iC3b generating fragments C3c (C3c alpha′ chainfragment1: amino acids 749-954 of SEQ ID NO: 9 and C3c alpha′ chainfragment 2: amino acids 1321-1663 of SEQ ID NO: 9) and C3dg (amino acids955-1303 of SEQ ID NO: 9). The end result is that C3b is permanentlyinactivated (see Sahu and Lambris (2001) Immunological Reviews180:35-48). C3dg can be further cleaved to generate fragments C3g (aminoacids 955-1001 of SEQ ID NO: 9) and C3d (amino acids 1002-1303 of SEQ IDNO: 9).

D. MODIFIED MTSP-1 POLYPEPTIDES THAT CLEAVE C3

Provided herein are modified or variant membrane type-serine protease 1(MTSP-1) polypeptides. The modified MTSP-1 polypeptides provided hereinexhibit altered activities or properties compared to a wild-type, nativeor reference MTSP-1 polypeptide. For example, the MTSP-1 polypeptidesprovided herein contain modifications compared to a wild-type, native orreference MTSP-1 polypeptide set forth in any of SEQ ID NOs:1-4, or in apolypeptide that has at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, particularlyat least 95%, sequence identity to any of SEQ ID NOs: 1-4, such as thereference MTSP-1 protease domain set forth in SEQ ID NO:4. Includedamong the modified MTSP-1 polypeptides provided herein are MTSP-1polypeptides that alter (inhibit) complement activation by effectinginhibitory cleavage of complement protein C3. Among the modified MTSP-1polypeptides provided herein are those that effect inhibitory cleavageof complement protein C3. Included are those that effect inhibitorycleavage of C3 with greater activity or specificity, k_(cat)/K_(m),compared to a corresponding form of the MTSP-1 that does not contain themodification (the replacement, deletion and/or insertion) or compared tothe corresponding form of unmodified MTSP-1 whose sequences are setforth in any of SEQ ID NOs:1-4. The modified MTSP-1 polypeptides alsocan have decreased specificity and/or and selectivity for substrates andtargets cleaved or recognized by unmodified MTSP-1 compared to a MTSP-1polypeptide not containing the amino acid modification(s), such as, forexample, proteinase-activated receptor-2 (PAR-2), urokinase-typeplasminogen activator (uPA), and/or hepatocyte growth factor (HGF).

The modified MTSP-1 polypeptides provided herein inhibit or inactivatecomplement through inhibitory or inactivation cleavage of complementprotein C3. The modified MTSP-1 polypeptides provided herein inhibit orinactivate complement by cleaving complement protein C3 at a cleavagesite that results in inhibition or inactivation of C3. Inactivation orinhibition cleavage of complement protein C3 can be at any sequence inC3 so long as the resulting cleavage of C3 results in inactivation orinhibition of activation of complement. Since the modified MTSP-1polypeptides provided herein inhibit complement activation, the modifiedMTSP-1 polypeptides do not effect cleavage of the zymogen form of C3 togenerate the C3a and C3b activated fragments. Thus, modified MTSP-1polypeptides provided herein do not cleave C3 between residues 748-749of SEQ ID NO: 9, which would result in generation of C3a and C3b.Inhibition or inactivation cleavage sites of complement protein C3 canbe empirically determined or identified. If necessary, a modified MTSP-1polypeptide provided herein can be tested for its ability to inhibitcomplement as described in section E below and as exemplified in theExamples.

The modified MTSP-1 polypeptides provided herein are isolated proteasedomains of MTSP-1. Smaller portions thereof that retain proteaseactivity also are contemplated. The protease domains provided herein aresingle-chain polypeptides with an N-terminus generated at the cleavagesite (generally having the consensus sequence R↓VVGG, R↓IVGG, R↓IVNG,R↓ILGG, R↓VGLL, R↓ILGG or a variation thereof; an N-terminus R↓V or R↓I,where the arrow represents the cleavage point) when the zymogen isactivated.

The protease domains generated herein, however, do not result fromactivation, which produces a two chain activated product, but rather aresingle chain polypeptides with the N-terminus including the consensussequence ↓VVGG, ↓IVGG, ↓VGLL, ↓ILGG or ↓IVNG or other such motif at theN-terminus. As shown herein, such polypeptides, although not the resultof activation and not double-chain forms, exhibit proteolytic(catalytic) activity. These protease domain polypeptides are used inassays to screen for agents that modulate the activity of the MT-SP.Such assays are also provided herein. In exemplary assays, the effectsof test compounds in the ability of a protease domain to proteolyticallycleave a known substrate, typically a fluorescently, chromogenically orotherwise detectably labeled substrate, are assessed. Agents, generallycompounds, particularly small molecules, that modulate the activity ofthe protease domain are candidate compounds for modulating the activityof the MT-SP. The protease domains can also be used to producesingle-chain protease-specific antibodies. The protease domains providedherein include, but are not limited to, the single chain region havingan N-terminus at the cleavage site for activation of the zymogen,through the C-terminus, or C-terminal truncated portions thereof thatexhibit proteolytic activity as a single-chain polypeptide in in vitroproteolysis assays, of any MT-SP family member, preferably from amammal, including and most preferably human, such as, for example,MTSP-1.

The modified MTSP-1 polypeptides provided herein are mutants of thesingle chain protease domain of MTSP-1, particularly modified MTSP-1polypeptides in which the Cys residue in the protease domain that isfree (i.e., does not form disulfide linkages with any other Cys residuein the protein) is substituted with another amino acid substitution,preferably with a conservative amino acid substitution or a substitutionthat does not eliminate the activity, such as, for example, substitutionwith Serine, and modified MTSP-1 polypeptides in which a glycosylationsite(s) is eliminated. Modified MTSP-1 polypeptides in which otherconservative amino acid substitutions in which catalytic activity isretained are also contemplated (see, e.g., Table 3, for exemplary aminoacid substitutions).

The modified MTSP-1 polypeptides provided herein catalyze inhibitory orinactivation cleavage of complement protein C3. The modified MTSP-1polypeptides provided herein cleave complement protein C3 at anycleavage sequence as long as the resulting C3 fragments are inactive, orunable to activate a complement-mediated effector function. The modifiedMTSP-1 polypeptides provided herein include those that have altered(i.e., decreased) specificity and/or selectivity for natural targets ofMTSP-1. In one example, the modified MTSP-1 polypeptides provided hereinhave reduced selectivity for PAR-2, uPA and/or HGF. In other examples,the modified MTSP-1 polypeptides provided herein have increasedspecificity for cleavage of complement protein C3, and decreasedspecificity for PAR-2, uPA and/or HGF.

The modified MTSP-1 polypeptides provided herein contain one or moreamino acid modifications such that they cleave complement protein C3 ina manner that results in inactivation or inhibition of complement. Themodifications can be a single amino acid modification, such as singleamino acid replacements (substitutions), insertions or deletions, ormultiple amino acid modifications, such as multiple amino acidreplacements, insertions or deletions. Exemplary modifications are aminoacid replacements, including single or multiple amino acid replacements.The amino acid replacement can be a conservative substitution, such asset forth in Table 3, or a non-conservative substitution, such as anydescribed herein. Modified MTSP-1 polypeptides provided herein cancontain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more modified positions compared to the MTSP-1polypeptide not containing the modification.

The modifications described herein can be made in any MTSP-1polypeptide. For example, the modifications are made in a human MTSP-1polypeptide having a sequence of amino acids including or set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; a mouse MTSP-1polypeptide having a sequence of amino acids including or set forth inSEQ ID NO:12; or a rat MTSP-1 polypeptide having a sequence of aminoacids including or set forth in SEQ ID NO:13; or in sequence variants orcatalytically active fragments that exhibit at least 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to any of SEQ ID NOs:1-4, 12 and 13.

The modified MTSP-1 polypeptides provided herein can be modified in anyregion or domain of a MTSP-1 polypeptide provided herein, as long as themodified MTSP-1 polypeptide retains its ability to effect inactivationor inhibitory cleavage of complement protein C3. The modified MTSP-1polypeptides provided herein can be single-chain or two chainpolypeptides, species variants, splice variants, allelic variants,isoforms, or catalytically active fragments thereof, such as, forexample, the protease domain thereof. The MTSP-1 polypeptides providedherein can be full length or truncated MTSP-1 polypeptides. The modifiedMTSP-1 polypeptides provided herein can be the protease domain of MTSP-1or a modified form of the protease domain of MTSP-1. Also contemplatedfor use herein are zymogen, precursor or mature forms of modified MTSP-1polypeptides, provided the MTSP-1 polypeptides retain their ability toeffect inhibitory or inactivation cleavage of complement protein C3.Modifications in a MTSP-1 polypeptide also can be made to a MTSP-1polypeptide that also contains other modifications, includingmodifications of the primary sequence and modifications not in theprimary sequence of the polypeptide. For example, modificationsdescribed herein can be in a MTSP-1 polypeptide that is a fusionpolypeptide or chimeric polypeptide. The modified MTSP-1 polypeptidesprovided herein also include polypeptides that are conjugated to apolymer, such as a PEG reagent.

For purposes herein, reference to positions and amino acids formodification, including amino acid replacement or replacements, hereinare with reference to the MTSP-1 polypeptide set forth in any of SEQ IDNOs:1-4. It is within the level of one of skill in the art to make anyof the modifications provided herein in another MTSP-1 polypeptide byidentifying the corresponding amino acid residue in another MTSP-1polypeptide, such as the MTSP-1 polypeptide set forth in any of SEQ IDNOs:1-4 or a variant thereof that exhibits at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to a MTSP-1 polypeptide set forth in any of SEQID NOs:1-4. Corresponding positions in another MTSP-1 polypeptide can beidentified by alignment of the MTSP-1 polypeptide with the referenceMTSP-1 polypeptide set forth in any of SEQ ID NOs:1-4. For purposes ofmodification (e.g., amino acid replacement), the corresponding aminoacid residue can be any amino acid residue, and need not be identical tothe residue set forth in any of SEQ ID NOs:1-4. Typically, thecorresponding amino acid residue identified by alignment with, forexample, residues in SEQ ID NO:4 is an amino acid residue that isidentical to SEQ ID NO:4, or is a conservative or semi-conservativeamino acid residue thereto. It is also understood that the exemplaryreplacements provided herein can be made at the corresponding residue ina MTSP-1 polypeptide, such as the protease domain of MTSP-1, so long asthe replacement is different than exists in the unmodified or referenceform of the MTSP-1 polypeptide, such as the protease domain of MTSP-1.Based on this description and the description elsewhere herein, it iswithin the level of one of skill in the art to generate a modifiedMTSP-1 polypeptide containing any one or more of the describedmutations, and test each for a property or activity as described herein.

The modified MTSP-1 polypeptides provided herein alter complementactivity by proteolysis-mediated inhibition or inactivation ofcomplement protein C3. The modified MTSP-1 provided herein can havedecreased specificity for a MTSP-1 substrate, such as, for example,proteinase-activated receptor-2 (PAR-2), urokinase-type plasminogenactivator (uPA), and/or hepatocyte growth factor (HGF). For example, themodified MTSP-1 polypeptides provided herein exhibit less than 100% ofthe wild type activity of a MTSP-1 polypeptide for cleavage ofproteinase-activated receptor-2 (PAR-2), urokinase-type plasminogenactivator (uPA), and/or hepatocyte growth factor (HGF), such as lessthan 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activityfor cleavage of proteinase-activated receptor-2 (PAR-2), urokinase-typeplasminogen activator (uPA), and/or hepatocyte growth factor (HGF) of awild type or reference MTSP-1 polypeptide, such as the correspondingpolypeptide not containing the amino acid modification. In anotherexample, the modified MTSP-1 polypeptides provided herein exhibit lessthan 100% of the wild type binding activity of a MTSP-1 polypeptide forPAR-2, uPA and/or HGF, such as less than 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, 10% or less of the activity for binding to PAR-2, uPA and/orHGF of a wild type or reference MTSP-1 polypeptide, such as thecorresponding polypeptide not containing the amino acid modification.

Also provided herein are nucleic acid molecules that encode any of themodified MTSP-1 polypeptides provided herein. Nucleic acid moleculesthat encode a single-chain protease domain or catalytically activeportion thereof also are provided. In some examples, the encodingnucleic acid molecules also can be modified to contain a heterologoussignal sequence to alter (e.g., increased) expression and secretion ofthe polypeptide. The modified MTSP-1 polypeptides and encoding nucleicacid molecules provided herein can be produced or isolated by any methodknown in the art including isolation from natural sources, isolation ofrecombinantly produced proteins in cells, tissues and organisms, and byrecombinant methods and by methods including in silico steps, syntheticmethods and any methods known to those of skill in the art. The modifiedpolypeptides and encoding nucleic acid molecules provided herein can beproduced by standard recombinant DNA techniques known to one of skill inthe art. Any method known in the art to effect mutation of any one ormore amino acids in a target protein can be employed. Methods includestandard site-directed or random mutagenesis of encoding nucleic acidmolecules, or solid phase polypeptide synthesis methods. For example,nucleic acid molecules encoding a MTSP-1 polypeptide can be subjected tomutagenesis, such as random mutagenesis of the encoding nucleic acid,error-prone PCR, site-directed mutagenesis, overlap PCR, gene shuffling,or other recombinant methods. The nucleic acid encoding the polypeptidescan then be introduced into a host cell to be expressed heterologously.Hence, also provided herein are nucleic acid molecules encoding any ofthe modified polypeptides provided herein. In some examples, themodified MTSP-1 polypeptides are produced synthetically, such as usingsolid phase or solution phase peptide synthesis.

The MTSP-1 polypeptides provided herein have been modified to haveincreased specificity and/or selectivity for cleavage of an inhibitoryor inactivation cleavage sequence of complement protein C3. MTSP-1polypeptides can be modified using any method known in the art formodification of proteins. Such methods include site-directed and randommutagenesis. Assays such as the assays for biological function ofcomplement activation provided herein and known in the art can be usedto assess the biological function of a modified MTSP-1 polypeptide todetermine if the modified MTSP-1 polypeptide targets complement proteinC3 for cleavage and inactivation. Exemplary methods to identify anMTSP-1 polypeptide and the modified MTSP-1 polypeptides are providedherein.

1. Exemplary Modified MTSP-1 Polypeptides

Provided herein are modified MTSP-1 polypeptides that contain one ormore, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and moreamino acid modifications in a MTSP-1 polypeptide and that cleavecomplement protein C3 such that complement is inhibited or inactivated.Modifications are in the primary amino acid sequence, and includereplacements, deletions and insertions of amino acid residues. Themodification alters the specificity/activity of the MTSP-1 polypeptide.The modified MTSP-1 polypeptides herein are designed or selected torecognize and cleave a target site in a complement protein, particularlyC3 in a site that inactivates C3. They also can be further modified andscreened to have reduced specificity/activity on in vivo naturalsubstrates and/or to cleave such substrates less than an unmodifiedwild-type MTSP-1 polypeptide. They can be selected and identified by anysuitable protease screening method. The modified MTSP-1 polypeptidesherein initially were identified using the screening method described inU.S. Pat. No. 8,211,428, in which a library of modified proteases arereacted with a cognate or other inhibitory serpin, such as ATIII that ismodified to include a target sequence in the reactive site loop tocapture modified proteases that would cleave such target.

Modified MTSP-1 polypeptides provided herein display increased activityor specificity or k_(cat)/K_(m) for complement protein C3 at a site thatinactivates C3, and also can have reduced activity or specificity and/ordisplay increased selectivity, specificity and/or activity for a targetsite on complement protein C3, whereby the modified MTSP-1 polypeptideinactivates C3. The modified MTSP-1 polypeptides exhibit increasedactivity for cleaving and inactivating C3 compared to the correspondingform of wild-type or wild-type with the replacement C122S (bychymotrypsin numbering). In particular, the protease domain of themodified polypeptide exhibits increased inactivation cleavage activityof C3 compared to the MTSP-1 protease domain of SEQ ID NO:4 (MTSP-1protease domain with C122S). The increase in activity can be 10%, 20%,50%, 100%, 1-fold, 2-fold, 3-fold, 4, 5, 6, 7, 8, 9, 10-fold and morecompared to the unmodified MTSP-1.

For example, the modified MTSP-1 polypeptide can exhibit 110-1000% ormore of the MTSP-1 activity of a wild type or reference MTSP-1polypeptide, such as the MTSP-1 polypeptide set forth in any of SEQ IDNOs:1-4 for inactivating C3. For example, modified MTSP-1 polypeptidesprovided herein exhibit 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% ormore of the activity of the unmodified or reference MTSP-1 polypeptide,such as the corresponding polypeptide not containing the amino acidmodification (e.g. amino acid replacement), for example, a MTSP-1protease domain set forth in any of SEQ ID NOs:1-4. For example,exemplary positions that can be modified, for example by amino acidreplacement or substitution, include, but are not limited to, any ofpositions corresponding to position 637, 640, 658, 661, 664, 666, 705,706, 707, 708, 731, 759, 783, or 802 with reference to the sequence ofamino acids set forth in SEQ ID NO:1 (corresponding to positions 38, 41,59, 60b, 60e, 60g, 96, 97, ins97a, 98, 99, 122, 151, 175, 192 accordingto chymotrypsin numbering). For example, the amino acid positions can bereplacements at positions corresponding to replacement of glutamine (Q)at position 637, I640, Y658, D661, F664, Y666, D705, F706, T707, F708,C731, G759, Q783, or Q802 with reference to amino acid positions setforth in SEQ ID NO:1 (corresponding to Q38, I41, Y59, D60b, F60e, Y60g,D96, F97, T98, F99, C122, G151, Q175, Q192 according to chymotrypsinnumbering).

Exemplary amino acid replacements at any of the above positions are setforth in Table 11. Reference to corresponding position in Table 11 iswith reference to positions set forth in SEQ ID NO:1. It is understoodthat the replacements can be made in the corresponding position inanother MTSP-1 polypeptide by alignment with the sequence set forth inSEQ ID NO:1, whereby the corresponding position is the aligned position.For example, the replacement can be made in the MTSP-1 protease domainwith the sequence set forth in SEQ ID NO: 2 or a reference MTSP-1protease domain with the sequence set forth in SEQ ID NO: 4. In someexamples, the amino acid replacement(s) can be at the correspondingposition in a MTSP-1 polypeptide as set forth in SEQ ID NO: 4 or avariant thereof having at least or at least about 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, particularly 95%, or more sequence identity thereto, solong as the resulting modified MTSP-1 polypeptide exhibits altered(i.e., enhanced) specificity towards complement protein C3 compared to areference MTSP-1 polypeptide. In one example, any one or more of thereplacements are in any of SEQ ID NOs:1-4, so long as the resultingmodified MTSP-1 polypeptide exhibits altered (i.e., enhanced)specificity towards complement protein C3 compared to a reference MTSP-1polypeptide, such as, for example, a reference MTSP-1 polypeptide setforth in any of SEQ ID NOs: 1-4.

TABLE 11 Active Variants Corresponding Position Corresponding Position(in SEQ ID NO: 1) (chymotrypsin numbering) Replacement 637  38 H 640  41S, R, A, D 658  59 F 661   60b T, V 664   60e S, R K 666   60g W 705  96K, V, Y, L, I, P, E 706  97 G, T, E, D, N, Y, W   97a Insertion of V, E,A, G, N 707  98 P, G, N 708  99 L 731 122 S 759 151 H, N 783 175 L 802191 D, E, T

Exemplary of amino acid modifications in the modified MTSP-1polypeptides provided herein include, but are not limited to,replacement with histidine (H) at a position corresponding to position38; S at a position corresponding to position 41; R at a positioncorresponding to position 41; A at a position corresponding to position41; D at a position corresponding to position 41; F at a positioncorresponding to position 59; T at a position corresponding to position60b; V at a position corresponding to position 60b; S at a positioncorresponding to position 60e; R at a position corresponding to position60e; K at a position corresponding to position 60e; W at a positioncorresponding to position 60g; K at a position corresponding to position96; V at a position corresponding to position 96; Y at a positioncorresponding to position 96; L at a position corresponding to position96; I at a position corresponding to position 96; P at a positioncorresponding to position 96; E at a position corresponding to position96; G at a position corresponding to position 97; T at a positioncorresponding to position 97; E at a position corresponding to position97; D at a position corresponding to position 97; N at a positioncorresponding to position 97; Y at a position corresponding to position97; W at a position corresponding to position 97; V at a positioncorresponding to position 97a; E at a position corresponding to position97a; A at a position corresponding to position 97a; G at a positioncorresponding to position 97a; N at a position corresponding to position97a; P at a position corresponding to position 98; G at a positioncorresponding to position 98; N at a position corresponding to position98; L at a position corresponding to position 99; S at a positioncorresponding to position 122; H at a position corresponding to position151; N at a position corresponding to position 151; L at a positioncorresponding to position 175; D at a position corresponding to position192; E at a position corresponding to position 192; T at a positioncorresponding to position 192, according to chymotrypsin numbering eachwith reference to the amino acid positions set forth in SEQ ID NOs:1 or3. S at a position corresponding to position 731 (122S at a positioncorresponding to position 731) replaces a free Cys to thereby reduce atendency for aggregation.

Exemplary modified MTSP-1 polypeptides containing amino acidmodifications are set forth in Table 12a below. Table 12b includesmature numbering for exemplary modified MTSP-1 polypeptides. TheSequence ID No. references an exemplary MTSP-1 protease domain thatcontains the recited replacements, which include the replacement atC122S to reduce or eliminate aggregation. C122 is a free cysteine, whichcan result in cross-linking among the protease polypeptides; thisreplacement, while advantageous, is optional. It is understood, that thereferenced (by SEQ ID NO.) protease domain is exemplary, and full-lengthand precursor molecules, as well as other catalytically active portionsof the protease domain, full-length and precursor polypeptide caninclude the recited replacements.

TABLE 12a Modified MTSP-1 Polypeptides SEQ ID Chymotrypsin numbering NOI41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/ 21 Q192EQ38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/ 22F99L/C122S/G151N/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/ 23F99L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/ 24C122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/ 25C122S/G151H/Q175L/Q192D Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/26 F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/ 27C122S/G151N/Q175L/Q192D Q38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/28 F99L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97Y/ins97aN/ 29T98G/F99L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/ 30T98P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96P/F97W/ins97aN/ 31T98G/F99L/C122S/G151N/Q175L/Q192EQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/ 32C122S/G151N/Q175L/Q192D Q38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97E/ins97aV/33 T98G/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96L/F97D/ins97aG/ 34T98N/F99L/C122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 35T98P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/ 36T98P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/ 37T98P/F99L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97G/ins97aV/T98P/ 38F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/ins97aV/T98P/ 39F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 40T98P/F99L/C122S/G151H/Q175L I41E/F99L/C122S/G151N/Q192T 41I41D/C122S/G151N/Q192T 42 I41S/F99L/C122S/G151N/Q192V 43I41E/F99L/C122S/G151N/Q192T 44 I41D/Y59F/D96E/F99L/C122S/G151N/Q192T 45I41D/Y59F/C122S/G151N/Q192T 46I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/ 47C122S/G151H/Q175L/Q192D Q38H/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/48 F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/ 49C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/Y60gW/D96K/F97G/ins97aV/T98P/F99L/ 50C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/D96K/F97G/ins97aV/T98P/F99L/ 51C122S/G151H/Q175L/Q192D Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/T98P/F99L/52 C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/F99L/ 53C122S/G151H/Q175L/Q192D Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/54 T98P/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 55T98P/F99L/C122S/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 56T98P/F99L/C122S/G151H/Q192DQ38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D 57I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192D 58Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/ 59 Q192DI41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D 63Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/ 64 Q192DQ38H/I41S/D60bY/D96K/F97G/ins97aV/T98P/F99L/C122S/ 65 Q192D/D217VI41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192G/D217V 66I41S/D60bY/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192D/ 67 D217VI41S/D96M/F97G/ins97aV/T98P/F99L/C122S/Q192G/D217V 68I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192V/D217I 69I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192H 70I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192N/D217V 71I41S/D60bY/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/ 72 Q192DQ38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192G/ 73 D217VI41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192V 74I41S/P49S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192G/ 75 D217VI41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/Q192N/ 76 D217VI41T/F97W/F99L/C122S/G151N/Q175M/Q192G/D217L 77I41G/F97L/F99L/C122S/Q175A/Q192T/D217V 78I41G/F97V/F99L/C122S/G151Q/Q175M/Q192A/D217L 79I41G/F97I/F99L/C122S/G151L/Q175M/Q192S/D217V 80I41G/F97S/F99L/C122S/G151N/Q175L/Q192G/D217I 81F97E/F99L/C122S/D217I/K224N 154 C122S/G193A 155 C122S/G193E 156D96_F97delinsWYY/T98P/F99L/C122S 157 F97D/F99L/C122S/Q192G 158H40R/I41H/F97D/F99L/C122S/Q192G 159 C122S/G151N/G193A 160H40R/I41H/C122S/G151N 161 H40R/I41H/F97D/C122S/G151N 162H40R/I41H/F97E/C122S 163 F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192E 164H40R/I41H/Y60gL/F97D/F99L/C122S/G151N/Q175M/ 165 D217I/K224SH40R/I41H/F97D/F99L/C122S/G151D/Q192G 166 H40R/I41H/F97D/F99L/Q192G 167H40R/I41H/Y60gH/F97D/F99L/C122S/G151N/Q175A/ 168 Q192H/D217I/K224RH40R/I41H/Y60gF/F97D/F99L/C122S/Q192G/D217M/ 169 K224RH40R/I41H/Y60gF/F97D/F99L/C122S/Q192G/D217R/K224A 170H40R/I41H/F97D/F99L/C122S/Q175L/Q192G/D217K/ 171 K224AH40R/I41H/F97D/F99L/C122S/Q175M/Q192G/D217V/ 172 K224YH40R/I41H/F97D/F99L/C122S/Q175K/Q192G/D217I/K224H 173H40R/I41H/F97D/F99L/C122S/Q175M/Q192G/D217S 174H40R/I41H/Y60gF/F97D/F99L/C122S/Q175M/Q192G/ 175 D217W/K224RH40R/I41H/Y60gN/F97D/F99L/C122S/G151N/Q175K/ 176 Q192S/D217S/K224LH40R/I41H/Y60gH/F97D/F99L/C122S/Q175M/Q192G/ 177 D217I/K224LH40K/I41L/Y60gF/F97D/F99L/C122S/G151N/Q175R 178H40R/I41H/Y60gL/F97D/F99L/C122S/G151N 179H40K/I41M/Y60gG/F97D/F99L/C122S/G151N/Q175R/ 180 Q192R/D217V/K224SH40K/I41M/Y60gF/F97D/F99L/C122S/G151N/Q175L/ 181 Q192DH40R/I41H/F97D/C122S/G151N/Q175M/Q192A/D217S/ 182 K224RH40R/I41H/Y60gH/F97D/F99L/C122S/Q175M/Q192G/ 183 D217I/K224RH40R/I41H/F97D/F99L/C122S/G151D/Q175M/Q192G/ 184 D217VH40R/I41H/F97D/F99L/C122S/G151N/Q175M/Q192A/ 185 D217N/K224RH40R/I41H/F97D/F99L/C122S/G151N/Q175L/Q192A/ 186 D217N/K224RH40K/I41M/F97D/F99L/C122S/G151N/Q175M/Q192D/ 187 D217N/K224RH40K/I41M/F97D/F99L/C122S/G151N/Q175L/Q192A/ 188 D217N/K224RH40R/I41H/F97D/F99L/C122S/Q175M/Q192D/D217N/ 189 K224RH40R/I41H/F97D/F99L/C122S/Q175M/D217N/K224R 190H40K/I41M/F97D/F99L/C122S/Q175M/Q192D/D217N/ 191 K224RH40K/I41M/F97D/F99L/C122S/G151N/Q175M/Q192A/ 192 D217N/K224RH40K/I41M/F97D/F99L/C122S/Q175M/D217N/K224R 193H40R/I41H/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192E 194H40R/I41H/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192G 195H40R/I41H/F97E/ins97aE/T98G/F99L/C122S/Q175L/Q192G 196H40R/I41H/F97D/F99L/C122S/G151N/Q192H 197H40R/I41H/F97D/F99L/C122S/G151N/L153R 198H40R/I41H/F97D/C122S/G151N/L153R/V202M 199H40R/I41H/F97D/F99L/C122S/G151N/Q192H/P232S 200H40R/I41H/F97D/ins97aE/T98G/F99L/C122S/Q175L/Q192G 201H40R/I41H/F97D/C122S/G151N/L153R 202H40K/I41M/F99L/C122S/T150A/G151R/Q192G 203H40R/I41H/F97D/C122S/G133D/G151N 204 I41R/F99L/C122S/Q192G 205H40R/I41H/F99L/C122S/G151K/Q192G 206I41R/ins97aE/F97T/T98G/F99L/C122S/G151E/Q175L/Q192E 207K86R/K110R/C122S/K134R/K157R/K224R/K239R 208H40R/I41H/K86R/F97D/K110R/C122S/K134R/G151N/ 209 K157R/K224R/K239RK86R/F97T/ins97aE/T98G/F99L/K110R/C122S/K134R/ 210K157R/Q175L/Q192E/K224R/K239RH40R/I41H/F97D/F99L/C122S/Q175R/Q192G/D217H/K224S 211H40R/I41H/F97D/F99L/C122S/Q192G/D217I/K224S 212H40R/I41H/F97D/F99L/C122S/Q192G/D217K/K224A 213H40R/I41H/F97D/F99L/C122S/Q175R/Q192G/D217E/K224R 214H40R/I41H/F97D/C122S/Q175R/Q192G/D217I/K224Q 215H40P/I41R/F99L/C122S/Q192G 216 H40P/I41R/F99L/C122S/G151K/Q192G 217H40R/I41H/F99L/C122S/G151E/Q192G 218I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/Q175L/Q192E 219I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/Q175T/Q192E 220I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/Q175T/ 221 Q192DI41R/ins97aE/F97T/T98G/F99L/C122S/G151E/Q175T/Q192D 222H40P/I41R/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E 223H40P/I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/Q175L/ 224 Q192EI41R/ins97aE/F97T/T98G/F99L/C122S/G151N/Q175L/Q192E 225I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/Q175L/ 226 Q192DI41R/ins97aE/F97T/T98G/F99L/C122S/G151N/Q175T/Q192E 227I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/Q175T/ 228 Q192DI41R/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E 229H40P/I41R/F99L/C122S/G151E/Q192G 230I41R/F97T/ins97aE/T98G/F99L/C122S/G151E/Q175T/Q192E 231I41R/F97T/ins97aE/T98G/F99L/C122S/G151N/Q175S/Q192E 232I41R/F97T/ins97aE/T98G/F99L/C122S/G151N/Q175I/Q192E 233H40R/I41H/Y60gF/F97D/F99L/C122S/Q175K/Q192G/ 234 D217R/K224QH40R/I41H/F97D/F99L/C122S/Q175L/Q192G/D217Q/K224R 235H40R/I41H/F97D/F99L/C122S/G151N/Q192N/D217L/K224R 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509I41G/F97S/F99L/C122S/G151N/Q175I/D217T 510I41G/F97S/F99L/C122S/G151N/Q175I/Q192T 511F97R/ins97aT/T98V/C122S/Q175M/Q192T/D217S 512F97V/ins97aH/T98R/F99L/C122S/Q175R/Q192G/D217S 513F97Q/F99L/C122S/Q175N/Q192V/D217G 514F97L/ins97aM/T98N/F99L/C122S/Q175T/Q192T/D217S 515F97S/ins97aN/T98G/F99M/C122S/Q175T/Q192T/D217S 516I41T/F97H/F99L/C122S/G151N/Q175H/Q192V/D217L 517I41R/F97T/F99L/C122S/G151N/Q175T/Q192T/D217I 518I41E/F97V/F99L/C122S/G151N/Q175L/Q192S/D217H 519I41D/F97R/F99L/C122S/G151Q/Q175R/Q192V/D217L 520I41E/F97R/F99L/C122S/G151Q/Q175G/Q192T/D217V 521I41D/F97T/F99L/C122S/G151N/Q175S/Q192T/D217A 522I41E/F97A/F99M/C122S/G151N/Q175R/Q192S/D217E 523I41G/F99L/C122S/Q175I/Q192T/D217T 524 I41G/F99L/C122S/G151N/Q192I/D217I525 I41G/F99L/C122S/G151N/Q175I/Q192I 526F97V/F99L/C122S/G151N/Q175L/Q192S/D217H 527I41E/F99L/C122S/G151N/Q175L/Q192S/D217H 528I41E/F97V/F99L/C122S/Q175L/Q192S/D217H 529I41E/F97V/F99L/C122S/G151N/Q192S/D217H 530I41E/F97V/F99L/C122S/G151N/Q175L/D217H 531I41E/F97V/F99L/C122S/G151N/Q175L/Q192S 532F97R/F99L/C122S/G151Q/Q175R/Q192V/D217L 533I41D/F99L/C122S/G151Q/Q175R/Q192V/D217L 534I41D/F97R/F99L/C122S/Q175R/Q192V/D217L 535I41D/F97R/F99L/C122S/G151Q/Q192V/D217L 536I41D/F97R/F99L/C122S/G151Q/Q175R/D217L 537I41D/F97R/F99L/C122S/G151Q/Q175R/Q192V 538I41E/F97R/F99L/C122S/G151Q/Q175G/Q192T 539I41D/F99L/C122S/G151N/Q175S/Q192T/D217A 540I41D/F97T/F99L/C122S/Q175S/Q192T/D217A 541I41D/F97T/F99L/C122S/G151N/Q192T/D217A 542I41D/F97T/F99L/C122S/G151N/Q175S/D217A 543I41D/F97T/F99L/C122S/G151N/Q175S/Q192T 544F97T/F99L/C122S/G151N/Q175S/Q192T/D217A 545I41S/F97Q/F99L/C122S/Q175W/Q192V/D217R 546I41G/F97L/F99L/C122S/G151N/Q192V/D217L 547I41G/F97A/F99L/C122S/G151N/Q175M/Q192S 548I41G/F97V/F99L/C122S/G151N/Q192T/D217V 549I41D/F97R/F99L/C122S/Q175L/Q192T/D217I 550I41E/F97S/F99L/C122S/Q175L/Q192V/D217A 551F97L/F99L/C122S/Q175K/Q192V/D217M 552 F97E/F99L/C122S/G151A/Q192V 553F97E/F99L/C122S/Q175H/Q192V/D217P 554F97R/ins97aI/T98P/C122S/Q175M/Q192V/D217I 555I41D/F99L/C122S/G151N/Q192T/D217A 556 I41D/F99L/C122S/G151N/Q175S/Q192I557 I41D/F97T/F99L/C122S/G151N/Q192T 558 I41D/F99L/C122S/G151N/Q192T 559I41D/F99L/C122S/Q175S/Q192T 560 I41D/F99L/C122S/Q192T 561Q38R/I41G/Y60gG/F99M/C122S/G151N/Q192R 562Q38K/I41G/Y60gG/F99L/C122S/G151N/Q192H 563Q38L/I41R/Y60gF/F99L/C122S/G151N/Q192A 564Q38K/I41D/Y60gG/F99L/C122S/G151N 565Q38R/I41R/Y60gF/F99L/C122S/G151N/Q192G 566Q38S/I41S/Y60gW/F99L/C122S/G151D/Q192T 567Q38K/I41G/Y60gW/F99L/C122S/G151N/Q192A 568Q38H/I41S/Y60gW/C122S/G151H/Q192A 569Q38K/I41S/Y60gW/F99L/C122S/G151N/Q192G 570Q38F/I41S/Y60gA/C122S/G151N/Q192R 571Q38R/I41S/Y60gW/F99L/C122S/G151N/Q192E 572Q38K/I41R/Y60gG/F99L/C122S/G151N/Q192G 573Q38R/I41R/F99L/C122S/G151N/Q192G 574Q38R/I41R/Y60gL/F99L/C122S/G151N/Q192G 575 I41E/C122S/G151N/Q175L/Q192A576 I41S/F99M/C122S/G151N/Q175L/Q192G 577I41E/F99L/C122S/G151N/Q175L/Q192A 578 I41S/F99L/C122S/G151H/Q175L/Q192V579 I41G/F99L/C122S/G151N/Q192A 580 I41S/F99M/C122S/G151N/Q175G/Q192R581 I41E/F99L/C122S/G151N/Q175R/Q192H 582I41S/F99M/C122S/G151N/Q175E/Q192R 583 I41E/F99L/C122S/G151N/Q192V 584I41E/F99L/C122S/G151N/Q192S 585 I41S/F99L/C122S/G151N/Q175P/Q192V 586I41E/F99L/C122S/G151N/Q175G/Q192T 587 I41S/C122S/G151N/Q175R/Q192R 588I41S/F99M/C122S/G151N/Q175P/Q192S 589 I41S/C122S/G151N/Q175D/Q192R 590I41E/F99L/C122S/G151N/Q175R/Q192T 591 I41G/F99L/C122S/G151N/Q175R/Q192A592 F99L/C122S/G151N/Q192I 593 I41D/F99L/C122S/G151N 594I41S/F99L/C122S/Q175P/Q192V 595 I41S/F99L/C122S/G151N/Q175P 596I41E/F99L/C122S/Q175G/Q192T 597 I41E/F99L/C122S/G151N/Q175G 598I41D/Y59F/F99L/C122S/G151N/Q192T/V213A 599I41D/G43A/F99L/C122S/G151N/Q192T/P232S/K239R 600I41D/G43A/D96E/F99L/C122S/G151N/Q192T 601I41D/D96E/F99L/C122S/G151N/Q192T 602 I41D/D96E/C122S/G151N/Q192T 603I41D/D96E/F99L/C122S/G151N/Q192T/D217E 604I41D/F99L/M117T/C122S/G151N/Q192T/A204D/D217E 605I41D/F99L/C122S/G151N/Q192T/D217E 606I41D/F99L/C122S/I136M/G151N/Q192T/D217L/K224R 607I41D/F60eI/D96E/F99L/C122S/G151N/Q192T 608 I41D/C122S/G151N/Q192T/D217E609 D96E/C122S/G151N/Q192T 610 Y59F/C122S/G151N/Q192T 611Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/F99L/ 612 C122S/G151H/Q175L/Q192DI41S/ins97aV/C122S/Q192D 613 I41S/F97L/F99L/C122S/Q192S 614I41T/F97R/ins97aV/T98L/C122S/Q192S 615 I41S/F97V/T98N/F99L/C122S/Q192S616 I41S/F97G/ins97aA/T98L/C122S/Q192A 617 I41S/F97D/F99L/C122S/Q192V618 I41E/F97L/F99L/C122S/Q192A 619 I41S/F97A/ins97aV/T98L/C122S/Q192A620 I41S/F97del/T98S/F99L/C122S/Q192S 621I41A/Y60gW/D96F/F97G/F99M/C122S/Q175W/Q192A 622I41G/Y60gW/F99L/C122S/Q175R/Q192S 623I41A/Y60gW/ins97aE/F99L/C122S/Q175M/Q192T 624I41T/ins97aA/F99Y/C122S/Q175L/Q192A 625I41A/ins97aY/F99L/C122S/Q175R/Q192H 626I41S/ins97aT/F99L/C122S/Q175R/Q192H 627I41S/Y60gW/ins97aN/F99L/C122S/Q175R/Q192T 628Q38H/I41S/D96S/ins97aK/C122S/G151N/Q192A 629Q38H/I41A/D96A/ins97aA/C122S/G151D/Q192T 630Q38H/I41S/D96Q/ins97aT/C122S/G151N/Q192A 631Q38H/I41T/D96M/ins97aA/C122S/G151D 632 Q38Y/I41A/D96I/ins97aQ/C122S 633Q38H/I41S/D96K/ins97aT/C122S/G151K/Q192A 634Q38W/I41S/D96R/ins97aA/C122S/G151N/Q192A 635Q38H/I41A/D96R/ins97aQ/C122S 636 Q38F/I41V/D96Q/ins97aT/C122S/G151D 637L33M/Q38F/I41S/D96A/ins97aW/C122S/G151N/Q192S 638Q38H/I41S/D96V/ins97aA/C122S/G151N/Q192A 639Q38H/I41T/D96K/ins97aL/C122S/G151N/Q192A 640Q38H/I41S/D96Q/ins97aA/C122S/Q192T 641Q38W/I41V/D96R/ins97aA/C122S/G151N 642Q38Y/I41T/D96M/ins97aS/C122S/G151N 643Q38H/I41S/D96K/ins97aS/C122S/G151P/Q192S 644Q38H/I41S/D96G/ins97aG/C122S/G151N/Q192A 645Q38H/I41S/D96K/ins97aD/C122S/G151N/Q192S 646I41S/D96E/ins97aG/C122S/G151Q/Q192A 647I41S/Y59F/ins97aV/C122S/G187D/Q192V/D217V 648A35V/I41S/Y59F/C122S/Q192D/D217V 649 I41S/F93L/ins97aV/C122S/Q192V/D217V650 I41S/S90P/ins97aV/C122S/Y146E/Q192N/D217V 651I41S/S90T/ins97aV/C122S/Q192N/D217V 652I41S/S90T/ins97aV/C122S/Q192V/D217V 653 I41S/Y59F/ins97aV/C122S/Q192G654 I41S/Y59F/F97S/ins97aV/S116Y/C122S/Q192G/D217V 655I41S/ins97aV/C122S/Q192G/Q209L 656Q38H/I41S/ins97aV/A112V/C122S/Q192A/Q209L 657I41S/ins97aV/C122S/Q192V/D217V 658 I41S/Y59F/ins97aV/C122S/Q192A 659I41A/F97G/ins97aM/T98L/C122S 660 I41G/F97E/F99L/C122S/Q192A 661I41S/F97V/ins97aV/T98P/C122S 662 I41S/T98S/F99L/C122S/Q192A 663I41S/F97Q/F99L/C122S/Q192S 664 I41G/F97L/F99L/C122S/Q192S 665I41S/F97G/ins97aA/T98P/C122S/Q192A 666 I41A/F97G/ins97aV/T98E/C122S 667I41A/F97S/ins97aA/C122S 668 I41A/F97W/T98S/F99L/C122S/Q192A 669I41L/N95D/D96T/F97W/F99L/C122S/Q192A 670I41T/Y60gL/N95D/D96F/F97S/F99L/C122S/Q175S/Q192A 671I41A/Y60gW/N95D/D96F/F97G/F99L/C122S/Q175H/Q192A 672I41A/Y60gW/F99L/C122S/Q175T/Q192A 673 Q38M/I41T/D96M/ins97aH/C122S/G151E674 Q38H/I41T/D96R/ins97aG/C122S/G151S 675I41S/D60bY/ins97aV/T98N/C122S/Q192H 676I41S/Y59F/D60bY/ins97aV/C122S/Q192G 677I41S/D60bY/ins97aV/A112V/C122S/Q192G/Q209L 678A35T/I41S/Y59F/ins97aV/C122S/Y146FN183A/Q192G/ 679 R235HI41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175H/ 680 Q192D141S/ins97aV/C122S/N164D/Q192G/R235H 681I41S/Y59F/ins97aV/C122S/Q192G/N223D 682I41S/ins97aV/C122S/N164D/Q192G/R235L 683I41S/Y59F/F97Y/ins97aV/C122S/Q192G 684I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192V 685I41S/F99L/C122S/G151N/Q175M/Q192G/D217V 686I41S/F97L/F99L/C122S/G151N/Q192G/D217V 687I41S/F97S/F99L/C122S/G151N/Q175L/Q192A/D217L 688I41G/F97R/F99L/C122S/G151N/Q175L/Q192S/D217V 689I41T/F97L/F99L/C122S/G151N/Q175S/Q192S/D217W 690I41D/F97T/F99M/C122S/Q192V/D217M 691

TABLE 12b Modified MTSP-1 Polypeptides SEQ ID Mature MTSP-1 numberingChymotrypsin numbering NO I640R/F706T/InsE/T707G/I41R/F97T/Ins97aE/T98G/ 21 F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802E Q192E Q637H/I640A/D661V/F664R/Q38H/I41A/D60bV/F60eR/ 22 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802D Q192D Q637H/I640A/D661T/F664K/Q38H/I41A/D60bT/F60eK/ 23 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802D Q192D Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 24 Y666W/F706D/InsV/T707P/Y60gW/F97D/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802E Q192E Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 25 Y666W/F706D/InsV/T707P/Y60gW/F97D/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640A/D661T/F664K/Q38H/I41A/D60bT/F60eK/ 26 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 27 Y666W/F706D/InsV/T707P/Y60gW/F97D/ins97aV/T98P/ F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802D Q192D Q637H/I640A/D661V/F664R/Q38H/I41A/D60bV/F60eR/ 28 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640A/D661V/F664R/Q38H/I41A/D60bV/F60eR/ 29 Y666W/D705I/F706Y/InsN/Y60gW/D96I/F97Y/ins97aN/ T707G/F708L/C731S/G759N/ T98G/F99L/C122S/G151N/Q783L/Q802D Q175L/Q192D Q637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/30 Y666W/D705K/F706D/InsA/ Y60gW/D96K/F97D/ins97aA/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640A/D661V/F664R/ Q38H/I41A/D60bV/F60eR/ 31Y666W/D705P/F706W/InsN/ Y60gW/D96P/F97W/ins97aN/T707G/F708L/C731S/G759N/ T98G/F99L/C122S/G151N/ Q783L/Q802E Q175L/Q192EQ637H/I640A/D661V/F664R/ Q38H/I41A/D60bV/F60eR/ 32Y666W/D705I/F706N/T707G/ Y60gW/D96I/F97N/T98G/F99L/F708L/C731S/G759N/Q783L/ C122S/G151N/Q175L/Q192D Q802DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 33Y666W/D705Y/F706E/InsV/ Y60gW/D96Y/F97E/ins97aV/T707G/F708L/C731S/G759H/ T98G/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 34Y666W/D705L/F706D/InsG/ Y60gW/D96L/F97D/ins97aG/T707N/F708L/C731S/G759H/ T98N/F99L/C122S/G151H/ Q783L/Q802E Q175L/Q192EQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 35Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 36Y666W/D705V/F706G/InsV/ Y60gW/D96V/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 37Y666W/D705K/F706D/InsA/ Y60gW/D96K/F97D/ins97aA/T707P/F708L/C731S/G759N/ T98P/F99L/C122S/G151N/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 38Y666W/F706G/InsV/T707P/ Y60gW/F97G/ins97aV/T98P/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 39Y666W/D705K/InsV/T707P/ Y60gW/D96K/ins97aV/T98P/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 40Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L Q175LI640E/F708L/C731S/G759N/ I41E/F99L/C122S/G151N/ 41 Q802T Q192TI640D/C731S/G759N/Q802T I41D/C122S/G151N/Q192T 42I640S/F708L/C731S/G759N/ I41S/F99L/C122S/G151N/ 43 Q802V Q192VI640E/F708L/C731S/G759N/ I41E/F99L/C122S/G151N/ 44 Q802T Q192TI640D/Y658F/D705E/F708L/ I41D/Y59F/D96E/F99L/C122S/ 45 C731S/G759N/Q802TG151N/Q192T I640D/Y658F/C731S/G759N/ I41D/Y59F/C122S/G151N/ 46 Q802TQ192T I640S/D661T/F664S/Y666W/ I41S/D60bT/F60eS/Y60gW/ 47D705K/F706G/InsV/T707P/ D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/D661T/F664S/Y666W/Q38H/D60bT/F60eS/Y60gW/ 48 D705K/F706G/InsV/T707P/D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/Q802D Q192D Q637H/I640S/F664S/Y666W/ Q38H/I41S/F60eS/Y60gW/ 49D705K/F706G/InsV/T707P/ D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640S/D661T/Y666W/Q38H/I41S/D60bT/Y60gW/ 50 D705K/F706G/InsV/T707P/D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/Q802D Q192D Q637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 51D705K/F706G/InsV/T707P/ D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 52 Y666W/D705K/F706G/T707P/ Y60gW/D96K/F97G/T98P/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 53Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 54Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/C731S/G759H/Q783L/ T98P/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 55Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/Q783L/ T98P/F99L/C122S/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 56Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q802D Q192DQ637H/I640S/D705K/F706G/ Q38H/I41S/D96K/F97G/ 57 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q802D Q192D I640S/D705K/F706G/InsV/I41S/D96K/F97G/ins97aV/ 58 T707P/F708L/C731S/Q783L/T98P/F99L/C122S/Q175L/ Q802D Q192D Q637H/I640S/D705K/F706G/Q38H/I41S/D96K/F97G/ 59 InsV/T707P/F708L/C731S/ ins97aV/T98P/F99L/C122S/Q783L/Q802D Q175L/Q192D I640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/63 T707P/F708L/C731S/Q802D T98P/F99L/C122S/Q192DQ637H/I640S/D705K/F706G/ Q38H/I41S/D96K/F97G/ 64 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661Y/D705K/ Q38H/I41S/D60bY/D96K/ 65F706G/InsV/T707P/F708L/ F97G/ins97aV/T98P/F99L/ C731S/Q802D/D828VC122S/Q192D/D217V I640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/ 66T707P/F708L/C731S/Q802G/ T98P/F99L/C122S/Q192G/ D828V D217VI640S/D661Y/D705K/F706G/ I41S/D60bY/D96K/F97G/ 67InsV/T707P/F708L/C731S/ ins97aV/T98P/F99L/C122S/ Q802D/D828V Q192D/D217VI640S/D705M/F706G/InsV/ I41S/D96M/F97G/ins97aV/ 68T707P/F708L/C731S/Q802G/ T98P/F99L/C122S/Q192G/ D828V D217VI640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/ 69T707P/F708L/C731S/Q802V/ T98P/F99L/C122S/Q192V/ D828I D217II640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/ 70T707P/F708L/C731S/Q802H T98P/F99L/C122S/Q192H I640S/D705K/F706G/InsV/I41S/D96K/F97G/ins97aV/ 71 T707P/F708L/C731S/Q802N/T98P/F99L/C122S/Q192N/ D828V D217V I640S/D661Y/D705K/F706G/I41S/D60bY/D96K/F97G/ 72 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D705K/F706G/ Q38H/I41S/D96K/F97G/ 73 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q802G/D828V Q192G/D217V I640S/D705K/F706G/InsV/I41S/D96K/F97G/ins97aV/ 74 T707P/F708L/C731S/Q783L/T98P/F99L/C122S/Q175L/ Q802V Q192V I640S/P648S/D705K/F706G/I41S/P49S/D96K/F97G/ 75 InsV/T707P/F708L/C731S/ ins97aV/T98P/F99L/C122S/Q802G/D828V Q192G/D217V I640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/76 T707P/F708L/C731S/Q783L/ T98P/F99L/C122S/Q175L/ Q802N/D828VQ192N/D217V I640T/F706W/F708L/C731S/ I41T/F97W/F99L/C122S/ 77G759N/Q783M/Q802G/ G151N/Q175M/Q192G/ D828L D217LI640G/F706L/F708L/C731S/ I41G/F97L/F99L/C122S/ 78 Q783A/Q802T/D828VQ175A/Q192T/D217V I640G/F706V/F708L/C731S/ I41G/F97V/F99L/C122S/ 79G759Q/Q783M/Q802A/D828L G151Q/Q175M/Q192A/D217L I640G/F706I/F708L/C731S/I41G/F97I/F99L/C122S/ 80 G759L/Q783M/Q802S/D828V G151L/Q175M/Q192S/D217VI640G/F706S/F708L/C731S/ I41G/F97S/F99L/C122S/ 81G759N/Q783L/Q802G/D828I G151N/Q175L/Q192G/D217I2. Additional Modifications

Any of the modified MTSP-1 polypeptides provided herein can contain anyone or more additional modifications. The additional modifications caninclude, for example, any amino acid substitution, deletion or insertionknown in the art, typically any that increase specificity of a modifiedMTSP-1 polypeptide for inactivation cleavage of complement protein C3compared to an unmodified or reference MTSP-1 polypeptide, such as theprotease domain. Any modified MTSP-1 polypeptide provided herein cancontain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 or more additional amino acid modifications. Also, contemplatedare modifications that alter any other activity of interest. It is longknown in the art that amino acid modifications of the primary sequenceare additive (see, e.g., Wells (1990) Biochem 29:8509-8517). Examples ofadditional modifications that can be included in the modified MTSP-1polypeptides provided herein include, but are not limited to, thosedescribed in U.S. Pat. Nos. 7,939,304; 8,211,428; 8,445,245; 9,290,757;9,359,598; 8,663,633; U.S. Patent Publication Nos. 2003/0119168;2007/0093443; 2010/0189652; 2010/0105121; 2012/0244139; 2014/0242062;2004/0146938; and 2009/0136477; Miyake et al. (2010) Biochim BioPhysActa 1804(1):156-165; List et al. (2007) J. Biol. Chem.282(50):36714-23; Desilets et al. (2008) J. Biol. Chem.283(16):10535-10542; Szabo (2009) Am J Pathol. 174(6):2015;Basel-Vanagaite et al. (2007) Am J Hum Genet. 80(3):467; Alef et al.(2009) J Invest Dermatol. 129(4):862; Ge et al. (2006) J. Biol. Chem.281:7406; Takeuchi et al. (1999) PNAS 96(20):11054-61; Oberst et al.(2003) J. Biol. Chem. 278(29):26773; and International PatentPublication Nos. WO 2015/166427 and WO 2015/085395. Non-limitingexamples of exemplary amino acid modifications described in the artinclude any one or more of R85H, N109Q, G149N, D251E, N302Q, Q348H,G349Y, R381S, P452R, D482Y, N485Q, D519Y, S524M, D555Y, C574R, D598Y,K600R, C602S, C604S, V615I, V616L, V616G, G617N, G617L, G618L, D622E,Q637D, I640T, I640A, I640L, I640F, I640D, I640E, C641S, L651M, H656A,C657S, D661I, D661F, D661R, D661A, R662F, R662D, R662A, R662W, Y666S,T673K, A674V, H679R, S685R, F702L, N704K, D705A, D705V, D705F, D7055,D705T, F706N, F706D, F706E, F706A, F706W, F706R, F706Y, F706L, T707P,F708Y, F708W, F708N, F708D, F708E, F708A, F708V, F708R, F7081, F708L,F708T, F708S, F708G, D711A, C731S, A735T, V738D, P740S, I745T, I745V,H752R, T753I, T755F, T755N, T755D, T755E, T755A, T755W, T755R, G756E,G759L, I762V, N772Q, N772D, T774A, T775P, C776S, L779F, L780N, L780D,L780E, L780A, L780V, L780F, L780R, P781S, Q780N, Q780D, Q780E, Q780A,Q780V, Q780F, Q780R, P781S, Q782H, Q782A, Q782V, Q782F, Q782R, Q782K,Q782L, Q782Y, Q783D, Q783E, Q783A, Q783V, Q783H, Q783H, Q783L, Q783F,Q783W, Q783Y, Q783R, Q783K, M788E, M788Y, M788R, M788A, C790S, F793L,C801S, Q802A, Q802V, Q802D, Q802R, Q802F, Q802X, Q802L, Q802I, Q802E,Q802K, Q802Y, Q802H, S805A, S811I, Q820L, W826F, W826Y, W826I, W826D,W826R, W826X, G827R, D828A, D828V, D828F, D828E, D828R, D828Q, D828N,D828H, C830S, Q832D, Q832L, Q832E, K835A, K835F, K835V, K835D, K835L,K835R, K835N, K835T, K835Y, K835S, K835F, R841W, F845L, and V855G,according to the sequence of amino acids set forth in SEQ ID NO:1.Additional modifications include amino acid replacements that introducea glycosylation site.

The modified MTSP-1 polypeptides include those that contain chemical orpost-translational modifications. In some examples, modified MTSP-1polypeptides provided herein do not contain chemical orpost-translational modifications. Chemical and post-translationalmodifications include, but are not limited to, PEGylation, sialylation,albumination, glycosylation, farnesylation, carboxylation,hydroxylation, phosphorylation, and other polypeptide modificationsknown in the art. Also, in addition to any one or more amino acidmodifications, such as amino acid replacements, provided herein,modified MTSP-1 polypeptides provided herein can be conjugated or fusedto any moiety using any method known in the art, including chemical andrecombinant methods, providing the resulting polypeptide retains theability to effect inhibitory or inactivation cleavage of complementprotein C3.

For example, in addition to any one or more amino acid modifications,such as amino acid replacements, provided herein, modified MTSP-1polypeptides provided herein also can contain other modifications thatare or are not in the primary sequence of the polypeptide, including,but not limited to, modification with a carbohydrate moiety, apolyethylene glycol (PEG) moiety, a sialylation moiety, an Fc domainfrom immunoglobulin G, or any other domain or moiety. For example, suchadditional modifications can be made to increase the stability or serumhalf-life of the protein.

a. Decreased Immunogenicity

The modified MTSP-1 polypeptides provided herein can be modified to havedecreased immunogenicity. Decreased immunogenicity can be effected bysequence changes that eliminate antigenic epitopes from the polypeptideor by altering post-translational modifications. One of skill in the artis familiar with methods of identifying antigenic epitopes in apolypeptide (see, e.g., Liang et al. (2009) BMC Bioinformatics, 10:302;Yang et al. (2009) Rev. Med. Virol., 19:77-96). In some examples, one ormore amino acids can be modified in order to remove or alter anantigenic epitope. In another example, altering the glycosylation of aprotein also can affect immunogenicity. For example, altering theglycosylation of the peptide is contemplated, so long as thepolypeptides retain the ability to effect inhibitory or inactivationcleavage of complement protein C3. Glycosylation sites can be removed bysingle mutations. Glycosylation sites can be added by introducing acanonical sequence, such as by insertion or single or a plurality ofmutations, such as NXS(T), where X is not a proline. Glycosylation sitesalso can increase serum half-life.

b. Fc Domains

The modified MTSP-1 polypeptides can be linked to the Fc region of animmunoglobulin polypeptide. Typically, such a fusion retains at least afunctionally active hinge, C_(H)2 and C_(H)3 domains of the constantregion of an immunoglobulin heavy chain. For example, a full-length Fcsequence of IgG1 includes amino acids 99-330 of the sequence set forthin the SEQ ID NO: 61. An exemplary Fc sequence for hIgG1 is set forth inSEQ ID NO: 62. It contains almost all of the hinge sequencecorresponding to amino acids 100-110 of SEQ ID NO:61; the completesequence for the C_(H)2 and C_(H)3 domain as set forth in SEQ ID NO:61.

Another exemplary Fc polypeptide is set forth in International PatentPublication No. WO 93/10151, and is a single chain polypeptide extendingfrom the N-terminal hinge region to the native C-terminus of the Fcregion of a human IgG1 antibody. The precise site at which the linkageis made is not critical: particular sites are well known and can beselected in order to optimize the biological activity, secretion, orbinding characteristics of the HABP polypeptide. For example, otherexemplary Fc polypeptide sequences begin at amino acid C109 or P113 ofthe sequence set forth in SEQ ID NO: 61 (see e.g., U.S. Pub. No.2006/0024298).

In addition to hIgG1 Fc, other Fc regions also can be used. For example,where effector functions mediated by Fc/FcγR interactions are to beminimized, fusion with IgG isotypes that poorly recruit complement oreffector cells, such as for example, the Fc of IgG2 or IgG4, iscontemplated. Additionally, the Fc fusions can contain immunoglobulinsequences that are substantially encoded by immunoglobulin genesbelonging to any of the antibody classes, including, but not limited toIgG (including human subclasses IgG1, IgG2, IgG3, or IgG4), IgA(including human subclasses IgA1 and IgA2), IgD, IgE, and IgM classes ofantibodies. Linkers can be used to covalently link Fc to anotherpolypeptide to generate a Fc chimera.

Modified Fc domains also are well known. In some examples, the Fc regionis modified such that it exhibits altered binding to an FcR resulting inaltered (i.e. more or less) effector function than the effector functionof an Fc region of a wild-type immunoglobulin heavy chain. Thus, amodified Fc domain can have altered affinity, including but not limitedto, increased or low or no affinity for the Fc receptor. For example,the different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4. In addition, different FcγRs mediatedifferent effector functions. FcγR1, FcγRIIa/c, and FcγRIIIa arepositive regulators of immune complex triggered activation,characterized by having an intracellular domain that has animmunoreceptor tyrosine-based activation motif (ITAM). FcγRIIb, however,has an immunoreceptor tyrosine-based inhibition motif (ITIM) and istherefore inhibitory. Altering the affinity of an Fc region for areceptor can modulate the effector functions and/or pharmacokineticproperties associated by the Fc domain. Modified Fc domains are known toone of skill in the art and described in the literature, see e.g., U.S.Pat. No. 5,457,035; U.S. Patent Publication No. US 2006/0024298; andInternational Patent Publication No. WO 2005/063816 for exemplarymodifications.

The resulting chimeric polypeptides containing Fc moieties, andmultimers formed therefrom, can be easily purified by affinitychromatography over Protein A or Protein G columns.

c. Conjugation to Polymers

In some examples, the modified MTSP-1 polypeptides provided herein areconjugated to polymers. Polymers can increase the size of thepolypeptide to reduce kidney clearance and thereby increase half-life orto modify the structure of the polypeptide to increase half-life orreduce immunogenicity. Exemplary polymers that can be conjugated to theMTSP-1 polypeptides, include natural and synthetic homopolymers, such aspolyols (i.e., poly-OH), polyamines (i.e., poly-NH₂) and polycarboxylicacids (i.e., poly-COOH), and other heteropolymers i.e. polymerscomprising one or more different coupling groups e.g. a hydroxyl groupand amine groups. Examples of suitable polymeric molecules includepolymeric molecules selected from among polyalkylene oxides (PAO), suchas polyalkylene glycols (PAG), including polyethylene glycols (PEG),methoxypolyethylene glycols (mPEG) and polypropylene glycols,PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG),branched polyethylene glycols (PEGs), polyvinyl alcohol (PVA),polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, dextrans including carboxymethyl-dextrans, heparin,homologous albumin, celluloses, including methylcellulose,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,carboxyethylcellulose and hydroxypropylcellulose, hydrolysates ofchitosan, starches such as hydroxyethyl-starches andhydroxypropyl-starches, glycogen, agaroses and derivatives thereof, guargum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acidhydrolysates and bio-polymers.

Typically, the polymers are polyalkylene oxides (PAO), such aspolyethylene oxides, such as PEG, typically mPEG, which have fewreactive groups capable of cross-linking. Typically, the polymers arenon-toxic polymeric molecules such as (methoxy)polyethylene glycol(mPEG) which can be covalently conjugated to the MTSP-1 polypeptides(e.g., to attachment groups on the protein surface) using a relativelysimple chemistry.

Suitable polymeric molecules for attachment to the MTSP-1 polypeptidesinclude, but are not limited to, polyethylene glycol (PEG) and PEGderivatives such as methoxy-polyethylene glycols (mPEG), PEG-glycidylethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGs,and polyethylene oxide (PEO) (see e.g., Roberts et al. (2002) AdvancedDrug Delivery Review 54: 459-476; Harris and Zalipsky (eds.)“Poly(ethylene glycol), Chemistry and Biological Applications” ACSSymposium Series 680, 1997; Mehvar et al. (2000) J. Pharm. Pharmaceut.Sci., 3(1):125-136; Harris and Chess (2003) Nat Rev Drug Discov.2(3):214-21; and Tsubery (2004), J Biol. Chem 279(37):38118-24). Thepolymeric molecule can be of a molecular weight typically ranging fromabout 3 kDa to about 60 kDa. In some embodiments the polymeric moleculethat is conjugated to a MTSP-1 polypeptide provided herein has amolecular weight of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 ormore than 60 kDa.

Methods of modifying polypeptides by covalently attaching (conjugating)a PEG or PEG derivative (i.e., “PEGylation”) are well known in the art(see e.g., U.S. 2006/0104968; U.S. Pat. Nos. 5,672,662; 6,737,505; andU.S. 2004/0235734). Techniques for PEGylation include, but are notlimited to, specialized linkers and coupling chemistries (see e.g.,Roberts et al., (2002) Adv. Drug Deliv. Rev. 54:459-476), attachment ofmultiple PEG moieties to a single conjugation site (such as via use ofbranched PEGs; see e.g., Guiotto et al. (2002) Bioorg. Med. Chem. Lett.12:177-180), site-specific PEGylation and/or mono-PEGylation (see e.g.,Chapman et al., (1999) Nature Biotech. 17:780-783), and site-directedenzymatic PEGylation (see e.g., Sato, Adv. Drug Deliv. Rev., 54:487-504,2002) (see, also, for example, Lu and Felix (1994) Int. J. PeptideProtein Res. 43:127-138; Lu and Felix (1993) Peptide Res. 6:140-146;Felix et al. (1995) Int. J. Peptide Res. 46:253-64; Benhar et al. (1994)J. Biol. Chem. 269:13398-404; Brumeanu et al. (1995) J Immunol.154:3088-95; see also, Caliceti et al. (2003) Adv. Drug Deliv. Rev.55(10):1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt 2):3S-8S).Methods and techniques described in the art can produce proteins having1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivativesattached to a single protein molecule (see e.g., U.S. 2006/0104968).

Numerous reagents for PEGylation have been described in the art. Suchreagents include, but are not limited to, N-hydroxysuccinimidyl (NHS)activated PEG, succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEGsuccinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEGsuccinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acidsuccinimidyl ester, homobifunctional PEG-succinimidyl propionate,homobifunctional PEG propionaldehyde, homobifunctional PEGbutyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl-carbonatePEG, mPEG-benzotriazole carbonate, propionaldehyde PEG, mPEGbutryaldehyde, branched mPEG2 butyraldehyde, mPEG acetyl, mPEGpiperidone, mPEG methylketone, mPEG “linkerless” maleimide, mPEG vinylsulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyldisulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylatePEG-NHS, fluorescein PEG-NHS, and biotin PEG-NHS (see e.g., Monfardiniet al., (1995) Bioconjugate Chem. 6:62-69; Veronese et al. (1997), J.Bioactive Compatible Polymers 12:196-207; U.S. Pat. Nos. 5,672,662;5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614;5,183,550; 5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581;5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237;6,113,906; 6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339;6,437,025; 6,448,369; 6,461,802; 6,828,401; and 6,858,736; U.S. PatentPublication Nos. 2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481;U.S. 2002/0052430; U.S. 2002/0072573; U.S. 2002/0156047; U.S.2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447;U.S. 2004/0013637; US 2004/0235734; U.S. 2005/0114037; U.S.2005/0171328; and U.S. 2005/0209416; European Patent Nos. EP 01064951and EP 0822199; and International Patent Publication Nos. WO 00/176640;WO 00/02017; WO 02/49673; WO 94/28024; and WO 01/87925).

d. Protein Transduction Domains

The modified MTSP-1 polypeptides provided herein can be linked, such asa fusion protein containing an antibody, or antigen binding fragmentthereof, conjugated to a protein transduction domain (PTD) thatincreases the retention of the antibody at a target site for therapy,such as a mucosal site, such as the eye. Any PTD can be employed so longas the PTD promotes the binding to target cell surfaces at thetherapeutic site (e.g. mucosal site) and/or uptake of the modifiedMTSP-1 polypeptide by target cells at the therapeutic site (e.g. mucosalsite, such as the eye).

Generally, PTDs include short cationic peptides that can bind to thecell surface through electrostatic attachment to the cell membrane andcan be uptaken by the cell by membrane translocation (Kabouridis (2003)TRENDS Biotech 21(11) 498-503). The PTDs provided generally interactwith a target cell via binding to glycosaminoglycans (GAGs), such as forexample, hyaluronic acid, heparin, heparan sulfate, dermatan sulfate,keratin sulfate or chondroitin sulfate and their derivatives.

The protein transduction domain can be of any length. Generally thelength of the PTD ranges from 5 or about 5 to 100 or about 100 aminoacids in length. For example, the length of the PTD can range from 5 orabout 5 to 25 or about 25 amino acids in length. In some examples, thePTD is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 or 25 amino acids in length.

A single PTD or a plurality thereof can be conjugated to a modifiedMTSP-1 polypeptide. These are advantageously employed for treatment ofocular or ophthalmic disorders, such as diabetic retinopathies ormacular degeneration, including AMD. For example, multiple copies of thesame PTD (e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer,octamer, nonamer, decamer or larger multimer) or different PTDs can beconjugated to the modified MTSP-1 polypeptide.

Several proteins and their peptide derivatives possess cellinternalization properties. Exemplary PTDs are known in the art andinclude, but are not limited to, PTDs listed in Table 13 below,including, for example, PTDs derived from human immunodeficiency virus 1(HIV-1) TAT (SEQ ID NOs:125-135; Ruben et al. (1989) J. Virol. 63:1-8),the herpes virus tegument protein VP22 (SEQ ID NO: 140; Elliott andO'Hare (1997) Cell 88:223-233), the homeotic protein of Drosophilamelanogaster Antennapedia (Antp) protein (Penetratin PTD; SEQ ID NO:112; Derossi et al. (1996) J. Biol. Chem. 271:18188-18193), theprotegrin 1 (PG-1) anti-microbial peptide SynB (e.g., SynB1 (SEQ ID NO:121), SynB3 (SEQ ID NO: 122), and Syn B4 (SEQ ID NO: 123); Kokryakov etal. (1993) FEBS Lett. 327:231-236) and the Kaposi fibroblast growthfactor (SEQ ID NO: 105; Lin et al. (1995) J. Biol. Chem.270-14255-14258). Other proteins and their peptide derivatives have beenfound to possess similar cell internalization properties. The carrierpeptides that have been derived from these proteins show little sequencehomology with each other, but are all highly cationic and arginine orlysine rich. Indeed, synthetic poly-arginine peptides have been shown tobe internalized with a high level of efficiency and can be selected forconjugation to an antibody provided (Futaki et al. (2003) J. Mol.Recognit. 16:260-264; Futaki et al. (2001) J. Biol. Chem.276:5836-5840). The PTD also can be selected from among one or moresynthetic PTDs, including but not limited to, transportan (SEQ ID NO:136; Pooga et al. (1998) FASEB J. 12:67-77; Pooga et al. (2001) FASEB J.15:1451-1453), MAP (SEQ ID NO: 103; Oehlke et al. (1998) Biochim.Biophys. Acta. 1414:127-139), KALA (SEQ ID NO: 101; Wyman et al. (1997)Biochemistry 36:3008-3017) and other cationic peptides, such as, forexample, various β-cationic peptides (Akkarawongsa et al. (2008)Antimicrob. Agents and Chemother. 52(6):2120-2129). Additional PTDpeptides and variant PTDs also are provided in, for example, U.S. PatentPublication Nos. US 2005/0260756, US 2006/0178297, US 2006/0100134, US2006/0222657, US 2007/0161595, US 2007/0129305, European PatentPublication No. EP 1867661, PCT Publication Nos. WO 2000/062067, WO2003/035892, WO 2007/097561, WO 2007/053512 and Table 13 herein (below).Any such PTDs provided herein or known in the art can be conjugated to aprovided therapeutic antibody.

TABLE 13 Known Protein Transduction Domains SEQ IDProtein Transduction Domain (PTD) Source Protein NOTRSSRAGLQFPVGRVHRLLRK Buforin II  82 RKKRRRESRKKRRRES DPV3  83GRPRESGKKRKRKRLKP DPV6  84 GKRKKKGKLGKKRDP DPV7  85 GKRKKKGKLGKKRPRSRDPV7b  86 RKKRRRESRRARRSPRHL DPV3/10  87 SRRARRSPRESGKKRKRKR DPV10/6  88VKRGLKLRHVRPRVTRMDV DPV1047  89 VKRGLKLRHVRPRVTRDV DPV1048  90SRRARRSPRHLGSG DPV10  91 LRRERQSRLRRERQSR DPV15  92GAYDLRRRERQSRLRRRERQSR DPV15b  93 WEAALAEALAEALAEHLAEALAEALEALAA GALA 94 KGSWYSMRKMSMKIRPFFPQQ Fibrinogen beta  95 chain KTRYYSMKKTTMKIIPFNRLFibrinogen gamma  96 chain precursor RGADYSLRAVRMKIRPLVTQFibrinogen alpha  97 chain LGTYTQDFNKFHTFPQTAIGVGAP hCT(9-32)  98TSPLNIHNGQKL FIN-1  99 NSAAFEDLRVLS Influenza virus 100nucleoprotein (NLS) WEAKLAKALAKALAKHLAKALAKALKACEA KALA 101 VPMLKPMLKEKu70 102 KLALKLALKALKAALKLA MAP 103 GALFLGFLGAAGSTMGAWSQPKKKRKV MPG 104AAVALLPAVLLALLAP Human Fibroblast 105 growth factor 4 (Kaposi Fibroblastgrowth factor) VQRKRQKLM N50 (NLS of NF-kB 106 P50)KETWWETWWTEWSQPKKKRKV Pep-1 107 SDLWEMMMVSLACQY Pep-7 108RQIKIWFQNRRMKWKK Penetratin 109 GRQIKIWFQNRRMKWKK Penetratin variant 110RRMKWKK Short Penetratin 111 ERQIKIWFQNRRMKWKK Penetratin 42-58 112RRRRRRR Poly Arginine-R7 113 RRRRRRRRR Poly Arginine-R9 114RVIRVWFQNKRCKDKK pISL 115 MANLGYWLLALFVTMWTDVGLCKKRPKPPrion mouse PrPc1- 116 28 LLIILRRRIRKQAHAHSK pVEC 117 LLIILRRRIRKQAHAHpVEC variant 118 VRLPPPVRLPPPVRLPPP SAP 119 PKKKRKV SV-40 (NLS) 120RGGRLSYSRRRFSTSTGR SynB1 121 RRLSYSRRRF SynB3 122 AWSFRVSYRGISYRRSRSynB4 123 YGRKKRRQRRRPPQ Tat 47-60 124 YGRKKRRQRRR Tat 47-57 125YGRKKRRQRR Tat 47-56 126 GRKKRRQRR Tat 48-56 127 GRKKRRQRRR Tat 48-57128 RKKRRQRRR Tat 49-57 129 RKKRRQRR Tat 49-56 130 GRKKRRQRRRPPQTat 48-60 131 GRKKR Tat 48-52 132 CFITKALGISYGRKKRRQRRRPPQFSQTHQVSLSKQTat 37-72 133 FITKALGISYGRKKRRQRRRPQFSQTHQVSLSKQ Tat 38-72 134YGRKKRRQRRRPP Tat 47-59 135 GWTLNSAGYLLGKINLKALAALAKKIL Transportan 136AGYLLGKINLKALAALAKKIL Transportan 10 137 GWTLNSAGYLLG Transportan 138derivative INLKALAALAKKIL Transportan 139 derivativeDAATATRGRSAASRPTERPRAPARSASRPRRPVD VP22 140 DPKGDPKGVTVTVTVTVTGKGDPKPDVT5 141 GALFLGWLGAAGSTMGAWSQPKKKRKV Signal Sequence- 142 based peptideKLALKLALKALKAALKLA Amphiphilic 143 model peptide KFFKFFKFFKBacterial cell wall 144 permeating LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTESLL-37 145 SWLSKTAKKLENSAKKRISEGIAIAIQGGPR Cecropin P1 146ACYCRIPACIAGERRYGTCIYQGRLWAFCC alpha defensin 147DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK beta defensin 148 RKCRIWIRVCRBactenecin 149 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRF PR-39 150 PGKRILPWKWPWWPWRR Indolicidin 151 GALFLGWLGAAGSTMGAWSQPKKKRKV MPS 152PVIRRVWFQNKRCKDKK pIs1 153

In some examples, the PTDs can be modified by replacement of a lysine orarginine with another basic amino acid, such as replacement of a lysinewith an arginine or by replacement of an arginine with a lysine.

E. ASSAYS TO ASSESS AND/OR MONITOR MTSP-1 ACTIVITY ONCOMPLEMENT-MEDIATED FUNCTIONS

The modified MTSP-1 polypeptides provided herein exhibit alteredspecificity and/or selectivity for complement protein C3. Exemplarymodified MTSP-1 polypeptides specifically cleave complement protein C3and thereby alter complement activation. Further, exemplary modifiedMTSP-1 polypeptides provided herein can have altered, or reduced,specificity and/or selectivity for cleavage of substrates of MTSP-1,such as, for example, proteinase-activated receptor-2 (PAR-2),urokinase-type plasminogen activator (uPA), and/or hepatocyte growthfactor (HGF).

Various in vitro and in vivo assays can be used to monitor or screenMTSP-1 polypeptides for their ability to cleave complement protein C3and for their effects on complement activation and complement-mediateddiseases and disorders. Such assays are well known to those of skill inthe art. One of skill in the art can test a particular MTSP-1polypeptide for cleavage of complement protein C3 and/or test to assessany change in the effects of a MTSP-1 on a complement-mediated activitycompared to the absence of a protease. Some such assays are exemplifiedherein.

Exemplary in vitro and in vivo assays are provided herein for comparisonof an activity of a modified MTSP-1 polypeptide on the function ofcomplement protein C3. In addition, numerous assays, such as assays formeasuring complement activation, are known to one of skill in the art.Assays for activities of complement include, but are not limited to,assays that measure activation products of complement activation, suchas for example the C5b-9 MAC complex, and generation of any one or moreof the complement cleavage products such as C5a, C3b, and C3d. Assays tomeasure complement activation also include functional assays thatmeasure the functional activity of specific components of the complementpathways, such as for example hemolytic assays used to measureactivation of any one of the classical, lectin or alternative pathways.Assays to assess effects of MTSP-1 polypeptides on complement proteinsand/or complement-mediated functions include, but are not limited to,SDS-PAGE analysis followed by Western Blot or Coomassie Brilliant Bluestaining, enzyme immunoassays, and hemolytic assays. In one example, invitro assays can be performed using purified complement protein C3, asexemplified in Example 2. In another example, in vivo assays can beperformed by testing the serum of a species, including mammalian orhuman species, for functional activation of complement, as exemplifiedin Examples 8 and 10. In another example, ex vivo assays can beperformed by testing the serum of a species, including mammalian orhuman species, for functional activation of complement, as exemplifiedin Examples 4-6. In another example, ex vivo assays can be performedusing peptide libraries, as exemplified in Examples 5-6. Various diseasemodels known to one of skill in the art can be used to test the efficacyof MTSP-1 polypeptides provided herein on various complement-mediateddiseases and disorders.

Also provided herein are exemplary assays for determining the activityof the modified MTSP-1 polypeptides for wild type MTSP-1 activities,such as cleavage of proteinase-activated receptor-2 (PAR-2),urokinase-type plasminogen activator (uPA), and/or hepatocyte growthfactor (HGF). Also provided are assays for determining the specificityof the modified MTSP-1 polypeptides for complement protein C3. Exemplaryassays are described below.

1. Methods for Assessing MTSP-1 Activity and Specificity for CleavingComplement Protein C3 to Inactivate it

A modified MTSP-1 protease can exhibit alterations in specificity and/orselectivity for inactivating C3, compared to the correspondingfull-length wildtype or protease domain or corresponding form of theunmodified MTSP-1 protease (i.e., the MTSP-1 polypeptides of SEQ IDNOs.: 1-4). Modified MTSP-1 proteases provided herein can retainprotease activity, but exhibit an increased specificity and/orselectivity or activity for cleaving C3 to thereby inhibit complementactivation. All such modified MTSP-1 proteases with increasedspecificity and/or selectivity to any one or more complement protein arecandidate therapeutics for treating any disorder in which complementactivation plays a role or is involved.

Where the modified MTSP-1 protease exhibits an increased specificityand/or selectivity to any one or more complement protein, in vitro andin vivo assays can be used to monitor or screen proteases for effects oncomplement-mediated functions. Such assays are well known to those ofskill in the art. One of skill in the art can test a modified MTSP-1protease for cleavage of C3 and/or test to assess any change in theeffects of a modified MTSP-1 protease on a complement-mediated activitycompared to the absence of a modified MTSP-1 protease. Some such assaysare exemplified herein.

Exemplary in vitro and in vivo assays are provided herein for comparisonof an activity of a modified MTSP-1 protease on the function of any oneor more targeted complement proteins. Many of the assays are applicableto other proteases and modified proteases. In addition, numerous assays,such as assays for measuring complement activation, are known to one ofskill in the art. Assays for activities of complement include, but arenot limited to, assays that measure activation products of complementactivation, such as for example the C5b-9 MAC complex, and generation ofany one or more of the complement cleavage products such as C4a, C5a,C3b, and C3d. Assays to measure complement activation also includefunctional assays that measure the functional activity of specificcomponents of the complement pathways, such as for example hemolyticassays used to measure activation of any one of the classical, lectin oralternative pathways. Assays to assess effects of proteases and modifiedproteases on complement proteins and/or complement-mediated functionsinclude, but are not limited to, SDS-analysis followed by Western Blotor Coomassie Brilliant Blue staining, enzyme immunoassays, and hemolyticassays. In one example, in vitro assays can be performed using purifiedcomplement proteins. In another example, in vivo assays can be performedby testing the serum of a species, including mammalian or human species,for functional activation of complement. Exemplary assays are describedbelow.

a. Protein Detection

Protein detection is a means to measure individual complement componentsin a sample. Complement proteins can be detected to assess directly theeffects of a MTSP-1 polypeptide on cleavage of complement protein C3, oralternatively, complement proteins can be measured as a means to assesscomplement activation. Complement protein C3, treated in the presence orabsence of a MTSP-1 polypeptide, can be analyzed by any one or moreassays including SDS-PAGE followed by Coomassie staining or WesternBlot, enzyme immunoassay, immunohistochemistry, flow cytometry,nephelometry, agar gel diffusion, or radial immunodiffusion. Exemplaryassays for protein detection are described below.

i. SDS-PAGE Analysis

Analysis of complement proteins in the presence or absence of increasingconcentrations of MTSP-1 polypeptide can be performed by analysis ofproteins on SDS-PAGE followed by detection of those proteins. In suchexamples, complement proteins can be detected by staining for totalprotein, such as by Coomassie Brilliant Blue stain, Silver stain, or byany other method known to one of skill in the art, or by Western Blotusing polyclonal or monoclonal antibodies specific for a specifiedprotein. Typically, a purified complement protein, such as for examplecomplement protein C3, can be incubated in the presence or absence of aMTSP-1 polypeptide. The treated complement protein can be resolved on anSDS-PAGE gel followed by a method to detect protein in the gel, forexample, by staining with Coomassie Brilliant blue. The treated proteincan be compared to its cognate full length protein and the degradationproducts formed by protease cleavage of the protein can be determined.

In another embodiment, a sample, such as for example human serum orplasma or breast milk, can be treated in the presence or absence of aMTSP-1 polypeptide or can be collected after treatment of an animal or ahuman with or without a MTSP-1 polypeptide. The MTSP-1-treated samplecan be analyzed on SDS-PAGE and a specific complement protein can bedetected, such as for example C3, C5, or Factor B, by Western Blot usingmonoclonal or polyclonal antibodies against the protein. The cleavage ofthe complement protein can be compared to a sample that was not treatedwith a MTSP-1 polypeptide. Additionally, the sample can be stimulated toinitiate complement activation such as by incubation with IgG whichstimulates activation of the classical pathway or by LPS whichstimulates activation of the alternative pathway. The sample can beresolved by SDS-PAGE for detection of any one or more of the nativecomplement proteins to determine the presence or absence of cleavageproducts of a specified protein compared to a sample of the protein nottreated with a MTSP-1 polypeptide. In such examples, cleavage effectormolecules of native complement proteins also can be analyzed by WesternBlot using monoclonal and polyclonal antibodies to assess the activationof one or more of the complement pathways. Examples of complementeffector molecules can include, but are not limited to, C3a, C3d, iC3b,Bb, and C5-b9. For example, decreased expression in a sample of Bb canindicate that a MTSP-1 polypeptide inhibited the activation of thealternative pathway of complement. The cleavage products of the effectormolecules also can be determined to assess the effects of increasingconcentrations of a MTSP-1 polypeptide on the cleavage of complementeffector molecules themselves.

ii. Enzyme Immunoassay

Enzyme immunoassay (EIA; also called enzyme-linked immunosorbent assay;ELISA) is an assay used to measure the presence of a protein in asample. Typically, measurement of the protein is an indirect measurementof the binding of the protein to an antibody, which itself is chemicallylabeled with a detectable substrate such as an enzyme or fluorescentcompound. EIAs can be used to measure the effects of MTSP-1 polypeptideson complement activation by measuring for the presence of a complementeffector molecule generated following complement activation. In suchexamples, a sample, such as for example human serum or plasma, can bepretreated in the presence or absence of increasing concentrations of aMTSP-1 polypeptide and subsequently activated to induce complementactivation by incubation with initiating molecules, or can be collectedfollowing treatment of an animal or a human with a MTSP-1 polypeptide.For example, the classical pathway can be activated by incubation withIgG and the alternative pathway can be activated by incubation of thesample with LPS. A complement activation assay specific for the lectinpathway requires that the classical pathway of complement is inhibitedsince the C4/C2 cleaving activity of the lectin pathway is shared withthe classical pathway of complement. Inhibition of the classical pathwaycan be achieved using a high ionic strength buffer which inhibits thebinding of C1q to immune complexes and disrupts the C1 complex, whereasa high ionic strength buffer does not affect the carbohydrate bindingactivity of MBL. Consequently, activation of the lectin pathway can beinduced by incubation of a sample, such as human serum or plasma, with amannan-coated surface in the presence of 1 M NaCl.

Following activation, the sample can be quenched with the addition ofPefabloc (Roche) and EDTA to minimize continued activation of thepathways. Samples can be analyzed for the presence of complementeffector molecules by an EIA or ELISA. EIAs and ELISAs for measuringcomplement proteins are well known to one skilled in the art. Anycomplement activation product can be assessed. Exemplary complementactivation products for measurement of complement activation includeiC3b, Bb, C5b-9, C3a, C3a-desArg and C5a-desArg. The complement pathwayactivated can be determined depending on the complement activationproduct measured. For example, measurement of Bb cleavage product is aunique marker of the alternative pathway.

In some examples, the EIA can be paired with detection of the cleavedcomplement proteins by analysis of the protease-treated,complement-stimulated sample by SDS-PAGE followed by Western blotanalysis for identification of specific complement components. Usingdensitometry software, the cleavage of the complement product can becompared to the full length complement component cleaved throughout theassay and the appearance of all major degradation products and thepercent cleavage can be determined.

iii. Radial Immunodiffusion (RID)

Radial immunodiffusion (RID) is a technique that relies on theprecipitation of immune complexes formed between antibodies incorporatedinto agarose gels when it is poured, and antigen present in a testsample resulting in a circular precipitin line around the sample well.The diameter of the precipitin ring is proportional to the concentrationof the antibody (or antigen) present in the test sample. By comparingthe diameter of the test specimen precipitin ring to known standards, arelatively insensitive estimation of the concentration of specificantibody or antigen can be achieved. RID can be used to measure theamount of a complement protein in a sample. For example, a sample suchas for example human serum or plasma, can be treated in the presence orabsence of increasing concentrations of a MTSP-1 polypeptide. Theprotease-treated sample can be added to a well of an agarose gel thathas been made to incorporate a polyclonal or monoclonal antibody againstany one of the complement proteins such as including, but not limitedto, C3, C5, C6, C7, C9, or Factor B. After removal of unprecipitatedproteins by exposure to 0.15 M NaCl, the precipitated protein rings canbe assessed by staining with a protein dye, such as for exampleCoomassie Brilliant blue or Crowles double stain.

b. Hemolytic Assays

Functional hemolytic assays provide information on complement functionas a whole. This type of assay uses antibody-sensitized or unsensitizedsheep erythrocytes. Hemolytic assays include the total hemolyticcomplement assay (CH50), which measures the ability of the classicalpathway and the MAC to lyse a sheep RBC. It depends on the sequentialactivation of the classical pathway components (C1 through C9) to lysesheep erythrocytes that have been sensitized with optimal amounts ofrabbit anti-sheep erythrocyte antibodies to make cellularantigen-antibody complexes. Hemolytic assays also can include analternative pathway CH50 assay (rabbit CH50 or APCH50), which measuresthe ability of the alternative pathway and the MAC to lyse a rabbit RBC.One CH50 and/or APCH50 unit is defined as the quantity or dilution ofserum required to lyse 50% of the red cells in the test. Typically, toassess complement activation, a sample, such as for example human serumor human plasma, can be treated in the presence or absence of increasingconcentrations of a MTSP-1 polypeptide, or can be collected followingtreatment of an animal or human in the presence or absence of a MTSP-1polypeptide. The protease-treated sample can be subsequently mixed withsheep's red blood cells that have been activated or sensitized with IgG.A water only sample mixed with sheep red blood cells can act as a totallysis control in order to accurately assess percent lysis of the samplesanalyzed. The addition of 0.15M NaCl to the sample can be added to stopthe lysing reaction. Lysis of the red blood cells, induced by theactivation of the terminal components of the complement pathway, can beassessed by measuring the release of hemoglobin. Measurement can be byoptical density (OD) readings of the samples using a spectrophotometerat an OD of 415 nm.

In one embodiment, limiting dilution hemolytic assays can be used tomeasure functional activity of specific components of either pathway. Insuch an assay, a serum source is used that has an excess of allcomplement components, but is deficient for the one being measured inthe sample, i.e., a media or serum source is complement-depleted for aspecific protein. The extent of hemolysis is therefore dependent on thepresence of the measured component in the test sample. In such an assay,a purified complement protein, such as for example any one of the nativecomplement proteins including, but not limited to C3, can be incubatedin the presence or absence of increasing concentrations of a MTSP-1polypeptide. The protease-treated purified complement protein can thenbe mixed with complement-depleted media or plasma and IgG-activatedsheep red blood cells and hemolysis of the sample can be assessed asdescribed above. In another embodiment, protease cleavage can becorrelated with complement activation by assaying for hemolytic activityof a protease-treated sample, and subsequently analyzing the sample onSDS-PAGE gel followed by staining with a protein stain, such as forexample Coomassie Blue. The purified complement protein treated with theproteases can be assessed for cleavage and the percentage of the fulllength complement component cleaved throughout the assay and theappearance of all major degradation products can be calculated.Alternatively, analysis of the protease-treated complement protein canbe by Western blot.

An alternative to the hemolytic assay, called the liposome immunoassay(LIA), can be used to assess activation of the classical pathway. TheLIA (Waco Chemicals USA, Richmond, Va.) utilizes dinitrophenyl(DNP)-coated liposomes that contain the enzyme glucose-6-phosphatedehydrogenase. When serum is mixed with the liposomes and a substratecontaining anti-DNP antibody, glucose-6-phosphate, and nicotinamideadenine dinucleotide, activated liposomes lyse, and an enzymaticcolorimetric reaction occurs which is proportional to total classicalcomplement activity.

c. Methods for Determining Cleavage Sites

Cleavage sequences in complement protein C3 can be identified by anymethod known in the art (see e.g., published U.S. Publication No. US2004/0146938). In one example, a cleavage sequence is determined byincubating complement protein C3 with any modified MTSP-1 polypeptideprovided herein. Following incubation with the MTSP-1 polypeptide, theC3 protein can be separated by SDS-PAGE and degradative products can beidentified by staining with a protein dye such as Coomassie BrilliantBlue. Proteolytic fragments can be sequenced to determine the identityof the cleavage sequences. After identification, fluorogenic peptidesubstrates designed based on the cleavage sequence of a desired targetsubstrate can be used to assess activity, as described below.

2. Methods for Assessing Wild Type MTSP-1 Activity

The modified MTSP-1 polypeptides provided herein can have altered, orreduced, specificity for their normal substrates, such as, for example,proteinase-activated receptor-2 (PAR-2), urokinase-type plasminogenactivator (uPA), and/or hepatocyte growth factor (HGF). Modified MTSP-1polypeptides can be tested to determine whether they retain catalyticefficiency and/or substrate specificity for their native substrate. Forexample, cleavage of PAR-2 can be assessed by incubation of a MTSP-1polypeptide with PAR-2 and detecting protein cleavage products. Inanother example, cleavage of PAR-2 can be determined in vitro bymeasuring cleavage of a fluorogenically tagged tetrapeptide of thepeptide substrate, for example, a fluorogenic substrate, such asfluorophores ACC (7-amino-4-carbamoyl-methy-coumarin)- orAMC-(7-amino-4-methylcoumarin) linked to a tetrapeptide. In someexamples, PAR-2 activation assays are used to determine the specificityof the MTSP-1 polypeptides provided herein.

In another example, cleavage of C3 can be assessed by incubation of aMTSP-1 polypeptide with C3 and detecting protein cleavage products. Inanother example, cleavage of C3 can be determined in vitro by measuringcleavage of a fluorogenically tagged tetrapeptide of the peptidesubstrate, for example, an ACC- or AMC-tetrapeptide. In some examples,C3 activation assays are used to determine the specificity of themodified MTSP-1 polypeptides provided herein. In another example,cleavage of HGF can be assessed by incubation of a modified MTSP-1polypeptide with HGF and detecting protein cleavage products. In anotherexample, cleavage of HGF can be determined in vitro by measuringcleavage of a fluorogenically tagged tetrapeptide of the peptidesubstrate, for example, an ACC- or AMC-tetrapeptide. In some examples,HGF activation assays are used to determine the specificity of theMTSP-1 polypeptides provided herein. In other examples, the ability ofthe MTSP-1 polypeptides provided herein to form a complex with theKunitz-type serine protease inhibitor, hepatocyte growth factoractivator inhibitor-1 (HAI-1) is determined.

a. Cleavage of MTSP-1 Substrates

In one example, modified MTSP-1 polypeptides can be assayed usingindividual fluorogenic peptide substrates corresponding to the desiredcleavage sequence. For example, a method of assaying for a modifiedMTSP-1 protease that can cleave any one or more of the desired cleavagesequences includes: (a) contacting a peptide fluorogenic sample(containing a desired target cleavage sequence) with a protease, in sucha manner whereby a fluorogenic moiety is released from a peptidesubstrate sequence upon action of the protease, thereby producing afluorescent moiety; and (b) observing whether the sample undergoes adetectable change in fluorescence, the detectable change being anindication of the presence of the enzymatically active protease in thesample. In such an example, the desired cleavage sequence is made into afluorogenic peptide by methods known in the art. In one embodiment, theindividual peptide cleavage sequences can be attached to afluorogenically tagged substrate, such as for example an ACC or AMCfluorogenic leaving group, and the release of the fluorogenic moiety canbe determined as a measure of specificity of a protease for a peptidecleavage sequence. The rate of increase in fluorescence of the targetcleavage sequence can be measured such as by using a fluorescencespectrophotometer. The rate of increase in fluorescence can be measuredover time. Michaelis-Menton kinetic constants can be determined by thestandard kinetic methods. The kinetic constants k_(cat), K_(m) andk_(cat)/K_(m) can be calculated by graphing the inverse of the substrateconcentration versus the inverse of the velocity of substrate cleavage,and fitting to the Lineweaver-Burk equation(1/velocity=(K_(m)/V_(max))(1/[S])+1/V_(max); whereV_(max)=[ET]k_(cat)). The second order rate constant or specificityconstant (k_(cat)/K_(m)) is a measure of how well a substrate is cut bya particular protease. For example, an ACC- or AMC-tetrapeptide such asAc-CPGR-AMC can be made and incubated with a modified MTSP-1 polypeptideprovided herein and activity of the MTSP-1 polypeptide can be assessedby assaying for release of the fluorogenic moiety. The choice of thetetrapeptide depends on the desired cleavage sequence to by assayed forand can be empirically determined.

In other embodiments, MTSP-1 polypeptides also can be assayed toascertain that they will cleave the desired sequence when presented inthe context of the full-length protein. In one example, a purifiedtarget protein, i.e., PAR-2, uPA or HGF, can be incubated in thepresence or absence of a selected MTSP-1 polypeptide and the cleavageevent can be monitored by SDS-PAGE followed by Coomassie Brilliant Bluestaining for protein and analysis of cleavage products usingdensitometry.

b. MTSP-1-Substrate Binding Assays

Binding of the MTSP-1 polypeptides to an MTSP-1 substrate can beassessed by any assay known to one of skill in the art to detectprotein-protein binding interactions, including but not limited to solidphase binding assays, ELISA, surface plasmon resonance and FACS. In oneexample, ELISA can be used. The recombinant substrate protein isimmobilized on a microtiter plate and MTSP-1 polypeptide binding ismeasured by addition of a reagent that specifically binds to MTSP-1,such as, for example, an MTSP-1 binding antibody. In another example,binding can be determined in a cell based assay using a cell line thatexpresses substrate. The MTSP-1 polypeptides can be labeled, forexample, with a chromogenic, fluorogenic or radioactive substrate toeffect detection of binding.

c. C3 Cleavage Assays

The activity of the modified MTSP-1 polypeptides can be assessed bycleavage of the substrate complement protein human C3 by measuring theamount of intact human C3 remaining after incubation with variousconcentrations of the modified MTSP-1 protease. In accord with thisassay, signal is generated in the presence of intact human C3, and islost as the C3 is cleaved.

Purified C3 protein can be incubated with the modified MTSP-1polypeptides and the residual levels of undigested human C3 can bequantified by any assay known in the art to assess proteinconcentration, such as, for example using an Amplified LuminescentProximity Homogeneous Assay Screen (AlphaScreen; Perkin Elmer). TheC3/MTSP-1 polypeptide mixture is incubated with α-mouse IgG-coatedacceptor beads, and following incubation the α-hC3 mAb/acceptor beadsmixture is incubated with a biotinylated α-hC3 pAb. Streptavidin-coateddonor beads are added to the mixture and the ‘alphascreen’ signal(Excitation=680 nm, Emission=570 nm) is then measured. This signalcorresponds to the concentration of remaining C3 protein. Theconcentration of MTSP-1 polypeptide required to cleave through 50% ofthe available hC3 (EC₅₀) can be calculated.

3. Specificity

The specificity constant of cleavage of target substrate, e.g.,complement protein C3 or an MTSP-1 substrate, such as, for example,PAR-2, uPA or HGF, by a modified MTSP-1 polypeptide can be determined byusing gel densitometry to assess changes in densitometry over time of afull-length target substrate incubated in the presence of a MTSP-1polypeptide. In specific embodiments, comparison of the specificities ofa modified MTSP-1 polypeptide can be used to determine if the modifiedMTSP-1 polypeptide exhibits altered, for example, increased, specificityfor C3 compared to the wild-type or reference MTSP-1 polypeptide. Thespecificity of a MTSP-1 polypeptide for a target substrate, e.g.complement protein C3, can be determined from the specificity constantof cleavage of a target substrate compared to a non-target substrate(e.g. the native wild type substrate of MTSP-1). A ratio of thespecificity constants of a modified MTSP-1 polypeptide for the targetsubstrate C3 versus a non-target substrate, such as, for example, PAR-2,uPA or HGF, can be made to determine a ratio of the efficiency ofcleavage of the modified MTSP-1 polypeptide. Comparison of the ratio ofthe efficiency of cleavage between a modified MTSP-1 polypeptide and awild-type or reference MTSP-1 polypeptide can be used to assess the foldchange in specificity for a target substrate. Specificity can be atleast 2-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 times or more when compared to thespecificity of a wild-type MTSP-1 polypeptide for a target substrateversus a non-target substrate.

Kinetic analysis of cleavage of native substrates of a MTSP-1polypeptide can be compared to analysis of cleavage of desired targetsubstrates in complement protein C3 to assess specificity of themodified MTSP-1 polypeptide for complement protein C3. In addition,second order rate constants of inhibition (ki) can be assessed tomonitor the efficiency and reactivity of a modified MTSP-1 polypeptidefor complement protein C3. For purposes herein, the modified MTSP-1polypeptides cleave C3 so that complement activation is inhibited, and,as shown in the Examples, they do so with significantly greateractivity, such as at least 5-fold more activity, than the unmodifiedMTSP-1 polypeptide (or modified MTSP-1 polypeptide with the C122Sreplacement, which eliminates a free cysteine to thereby reduceaggregation). For example, the modified MTSP-1 polypeptide of SEQ IDNO:35, cleaves human C3 in the assay described herein with an ED50 of 2nM, compared to 11 nM for the wild-type protease domain of SEQ ID NO:4.

4. Disease Models

The modified MTSP-1 polypeptides provided herein can be used in anyclinically relevant disease model known to one of skill in the art todetermine their effects on complement-mediated diseases or disorders.Exemplary assays include, but are not limited to, assays fortransplantation, including in vitro assays with human islet cells(Tjernberg et al. (2008) Transplantation 85:1193-1199) and ex vivoassays with pig kidneys (Fiane et al. (1999) Xenotransplantation6:52-65); bioincompatibility, including in vitro artificialsurface-induced inflammation (Lappegard et al. (2008) J Biomed Mater ResA 87:129-135; Lappegard et al. (2005) Ann Thorac Surg 79:917-923;Nilsson et al. (1998) Blood 92:1661-1667; Schmidt et al. (2003) J BiomedMater Res A 66:491-499); inflammation, including in vitro E.coli-induced inflammation (Mollnes et al. (2002) Blood 100:1869-1877)and heparin/protamine complex-induced inflammation in baboons (Soulikaet al. (2000) Clin Immunol 96:212-221); age-related macular degenerationin rabbits and monkeys and rodents (Chi et al. (2010) Adv Exp Med Biol703:127-135; Pennesi et al. (2012) Mol. Aspects Med. 33(4):487-509;Fletcher et al. (2014) Optm. Vis. Sci. 91(8):878-886; Forest et al.(2015) Disease Models and Mechanisms 8:421-427); and delayed graftfunction in pigs (Hanto et al., (2010) Am J Transplant 10(11):2421-2430)and dogs (Petrinec et al. (1996) Surgery 120:221-225).

F. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING MODIFIED MTSP-1POLYPEPTIDES

Polypeptides of a modified MTSP-1 polypeptide set forth herein can beobtained by methods well known in the art for protein purification andrecombinant protein expression. Polypeptides also can be synthesizedchemically. Modified or variant, including truncated forms, can beengineered from a wild type polypeptide using standard recombinant DNAmethods. For example, modified MTSP-1 polypeptides can be engineeredfrom a wild type polypeptide, such as by site-directed mutagenesis.

1. Isolation or Preparation of Nucleic Acids Encoding MTSP-1Polypeptides

Polypeptides can be cloned or isolated using any available methods knownin the art for cloning and isolating nucleic acid molecules. Suchmethods include PCR amplification of nucleic acids and screening oflibraries, including nucleic acid hybridization screening,antibody-based screening and activity-based screening. For example, whenthe polypeptides are produced by recombinant means, any method known tothose of skill in the art for identification of nucleic acids thatencode desired genes can be used. Any method available in the art can beused to obtain a full length or partial (i.e., encompassing the entirecoding region) cDNA or genomic DNA clone encoding a MTSP-1, such as froma cell or tissue source.

Methods for amplification of nucleic acids can be used to isolatenucleic acid molecules encoding a desired polypeptide, including forexample, polymerase chain reaction (PCR) methods. Exemplary of suchmethods include use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp). A nucleic acid containing material can be used asa starting material from which a desired polypeptide-encoding nucleicacid molecule can be isolated. For example, DNA and mRNA preparations,cell extracts, tissue extracts, fluid samples (e.g., blood, serum,saliva, breast milk), samples from healthy and/or diseased subjects canbe used in amplification methods. The source can be from any eukaryoticspecies including, but not limited to, vertebrate, mammalian, human,porcine, bovine, feline, avian, equine, canine, and other primatesources. Nucleic acid libraries also can be used as a source of startingmaterial. Primers can be designed to amplify a desired polypeptide. Forexample, primers can be designed based on expressed sequences from whicha desired polypeptide is generated. Primers can be designed based onback-translation of a polypeptide amino acid sequence. If desired,degenerate primers can be used for amplification. Oligonucleotideprimers that hybridize to sequences at the 3′ and 5′ termini of thedesired sequence can be used as primers to amplify by PCR sequences froma nucleic acid sample. Primers can be used to amplify the entirefull-length MTSP-1, or a truncated sequence thereof, such as a nucleicacid encoding any of the soluble MTSP-1 polypeptides provided herein.Nucleic acid molecules generated by amplification can be sequenced andconfirmed to encode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art. Additional nucleotide sequences suchas sequences of bases specifying protein binding regions also can belinked to enzyme-encoding nucleic acid molecules. Such regions include,but are not limited to, sequences of nucleotides that facilitate orencode proteins that facilitate uptake of an enzyme into specific targetcells, or otherwise alter pharmacokinetics of a product of a syntheticgene.

In addition, tags or other moieties can be added, for example, to aid indetection or affinity purification of the polypeptide. For example,additional nucleotide residues sequences such as sequences of basesspecifying an epitope tag or other detectable marker also can be linkedto enzyme-encoding nucleic acid molecules. Exemplary of such sequencesinclude nucleic acid sequences encoding a SUMO tag or His tag or FlagTag.

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, CA). The insertion into a cloning vector can, forexample, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. Insertion can beeffected using TOPO cloning vectors (Invitrogen, Carlsbad, CA).

If the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can contain specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and protein genecan be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells via, forexample, transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated. Inspecific embodiments, transformation of host cells with recombinant DNAmolecules that incorporate the isolated protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

In addition to recombinant production, modified MTSP-1 polypeptidesprovided herein, can be produced by direct peptide synthesis usingsolid-phase techniques (see e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, WH Freeman Co., San Francisco; Merrifield R. B.(1963) J Am Chem Soc., 85:2149-2154). In vitro protein synthesis can beperformed using manual techniques or by automation. Automated synthesiscan be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer, Foster City CA) in accordance with theinstructions provided by the manufacturer. Various fragments of apolypeptide can be chemically synthesized separately and combined usingchemical methods.

2. Generation of Mutant or Modified Nucleic Acids and EncodingPolypeptides

The modifications provided herein can be made by standard recombinantDNA techniques such as are routine to one of skill in the art. Anymethod known in the art to effect mutation of any one or more aminoacids in a target protein can be employed. Methods include standardsite-directed mutagenesis (using e.g., a kit, such as QuikChange®available from Stratagene) of encoding nucleic acid molecules, or bysolid phase polypeptide synthesis methods.

3. Vectors and Cells

For recombinant expression of one or more of the desired proteins, suchas any modified MTSP-1 polypeptide described herein, the nucleic acidcontaining all or a portion of the nucleotide sequence encoding theprotein can be inserted into an appropriate expression vector, i.e., avector that contains the necessary elements for the transcription andtranslation of the inserted protein coding sequence. The necessarytranscriptional and translational signals also can be supplied by thenative promoter for enzyme genes, and/or their flanking regions.

Also provided are vectors that contain a nucleic acid encoding theenzyme. Cells containing the vectors also are provided. The cellsinclude eukaryotic and prokaryotic cells, and the vectors are anysuitable for use therein. Generally, the cell is a cell that is capableof effecting glycosylation of the encoded protein.

Prokaryotic and eukaryotic cells containing the vectors are provided.Such cells include bacterial cells, yeast cells, fungal cells, Archea,plant cells, insect cells and animal cells. The cells are used toproduce a protein thereof by growing the above-described cells underconditions whereby the encoded protein is expressed by the cell, andrecovering the expressed protein. For purposes herein, for example, theenzyme can be secreted into the medium.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the polypeptide include,but are not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingcan impact the folding and/or function of the polypeptide. Differenthost cells, such as, but not limited to, CHO (DG44, DXB11, CHO-K1),HeLa, MCDK, 293 and WI38 have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and canbe chosen to ensure the correct modification and processing of theintroduced protein. Generally, the choice of cell is one that is capableof introducing N-linked glycosylation into the expressed polypeptide.Hence, eukaryotic cells containing the vectors are provided. Exemplaryof eukaryotic cells are mammalian Chinese Hamster Ovary (CHO) cells. Forexample, CHO cells deficient in dihydrofolate reductase (e.g., DG44cells) are used to produce polypeptides provided herein.

Provided are vectors that contain a sequence of nucleotides that encodesthe modified MTSP-1 polypeptide, such as the modified MTSP-1 proteasedomain, coupled to the native or heterologous signal sequence, as wellas multiple copies thereof. The vectors can be selected for expressionof the enzyme protein in the cell or such that the enzyme protein isexpressed as a secreted protein.

In one embodiment, vectors containing a sequence of nucleotides thatencodes a polypeptide that has protease activity and contains all or aportion of the protease domain, or multiple copies thereof, areprovided. Also provided are vectors that contain a sequence ofnucleotides that encodes the protease domain and additional portions ofa protease protein up to and including a full length protease protein,as well as multiple copies thereof. The vectors can be selected forexpression of the scaffold or modified protease protein or proteasedomain thereof in the cell or such that the protease protein isexpressed as a secreted protein. When the protease domain is expressedthe nucleic acid is linked to nucleic acid encoding a secretion signal,such as the Saccharomyces cerevisiae α-mating factor signal sequence ora portion thereof, or the native signal sequence.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include but are not limited to the SV40 early promoter(Benoist and Chambon (1981) Nature 290:304-310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.(1980) Cell 22:787-797), the herpes thymidine kinase promoter (Wagner etal. (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al. (1982) Nature296:39-42); prokaryotic expression vectors such as the β-lactamasepromoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:5543) or thetac promoter (DeBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25);see also “Useful Proteins from Recombinant Bacteria”: in ScientificAmerican 242:79-94 (1980); plant expression vectors containing thenopaline synthetase promoter (Herrara-Estrella et al. (1984) Nature303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardneret al. (1981) Nucleic Acids Res. 9:2871), and the promoter of thephotosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al. (1984) Nature 310:115-120); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al. (1984) Cell 38:639-646; Ornitz etal. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald(1987) Hepatology 7:42S-51S); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al. (1985) Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al. (1984) Cell 38:647-658; Adams et al.(1985) Nature 318:533-538; Alexander et al. (1987) Mol. Cell Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al. (1986) Cell45:485-495), albumin gene control region which is active in liver(Pinckert et al. (1987) Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al. (1985)Mol. Cell. Biol. 5:1639-1648; Hammer et al. (1987) Science 235:53-58),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al. (1987) Genes and Devel. 1:161-171), beta globin gene controlregion which is active in myeloid cells (Magram et al., (1985) Nature315:338-340; Kollias et al. (1986) Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells of thebrain (Readhead et al. (1987) Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Shani (1985),Nature 314:283-286), and gonadotrophic releasing hormone gene controlregion which is active in gonadotrophs of the hypothalamus (Mason et al.(1986) Science 234:1372-1378).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding a desired protein, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Depending on the expression system,specific initiation signals also are required for efficient translationof a MTSP-1 sequence. These signals include the ATG initiation codon andadjacent sequences. In cases where the initiation codon and upstreamsequences of MTSP-1 or catalytically active fragments thereof areinserted into the appropriate expression vector, no additionaltranslational control signals are needed. In cases where only codingsequence, or a portion thereof, is inserted, exogenous transcriptionalcontrol signals including the ATG initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading frameto ensure transcription of the entire insert. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression can be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf etal. (1994) Results Probl Cell Differ 20:125 62; Bitter et al. (1987)Methods in Enzymol 153:516-544).

Exemplary plasmid vectors for transformation of E. coli cells, include,for example, the pQE expression vectors (available from Qiagen,Valencia, CA; see also literature published by Qiagen describing thesystem). pQE vectors have a phage T5 promoter (recognized by E. coli RNApolymerase) and a double lac operator repression module to providetightly regulated, high-level expression of recombinant proteins in E.coli, a synthetic ribosomal binding site (RBS II) for efficienttranslation, a 6×His tag coding sequence, t₀ and T1 transcriptionalterminators, ColE1 origin of replication, and a beta-lactamase gene forconferring ampicillin resistance. The pQE vectors enable placement of a6×His tag at either the N- or C-terminus of the recombinant protein.Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiplecloning sites for all three reading frames and provide for theexpression of N-terminally 6×His-tagged proteins. Other exemplaryplasmid vectors for transformation of E. coli cells, include, forexample, the pET expression vectors (see, U.S. Pat. No. 4,952,496;available from Novagen, Madison, WI; see, also literature published byNovagen describing the system). Such plasmids include pET 11a, whichcontains the T7lac promoter, T7 terminator, the inducible E. coli lacoperator, and the lac repressor gene; pET 12a-c, which contains the T7promoter, T7 terminator, and the E. coli ompT secretion signal; and pET15b and pET19b (Novagen, Madison, WI), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

Typically, vectors can be plasmid, viral, or others known in the art,used for expression of the modified MTSP-1 polypeptide in vivo or invitro. For example, the modified MTSP-1 polypeptide is expressed inmammalian cells, including, for example, Chinese Hamster Ovary (CHO)cells.

Viral vectors, such as adenovirus, retrovirus or vaccinia virus vectors,can be employed. In some examples, the vector is a defective orattenuated retroviral or other viral vector (see U.S. Pat. No.4,980,286). For example, a retroviral vector can be used (see, Miller etal. (1993) Meth. Enzymol. 217: 581-599). These retroviral vectors havebeen modified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. Insome examples, viruses armed with a nucleic acid encoding a modifiedMTSP-1 polypeptide can facilitate their replication and spread within atarget tissue. The virus can also be a lytic virus or a non-lytic viruswherein the virus selectively replicates under a tissue specificpromoter. As the viruses replicate, the coexpression of the MTSP-1polypeptide with viral genes will facilitate the spread of the virus invivo.

4. Expression

Modified MTSP-1 polypeptides can be produced by any method known tothose of skill in the art including in vivo and in vitro methods. Themodified MTSP-1 polypeptides can be expressed in any organism suitableto produce the required amounts and forms of the proteins, such as forexample, needed for administration and treatment. Expression hostsinclude prokaryotic and eukaryotic organisms such as E. coli, yeast,plants, insect cells, mammalian cells, including human cell lines andtransgenic animals. Expression hosts can differ in their proteinproduction levels as well as the types of post-translationalmodifications that are present on the expressed proteins. The choice ofexpression host can be made based on these and other factors, such asregulatory and safety considerations, production costs and the need andmethods for purification. The skilled person is well-equipped to selectappropriate hosts and vectors.

Many expression vectors are available and known to those of skill in theart and can be used for expression of proteins. The choice of expressionvector will be influenced by the choice of host expression system. Ingeneral, expression vectors can include transcriptional promoters andoptionally enhancers, translational signals, and transcriptional andtranslational termination signals. Expression vectors that are used forstable transformation typically have a selectable marker which allowsselection and maintenance of the transformed cells. In some cases, anorigin of replication can be used to amplify the copy number of thevector.

Modified MTSP-1 polypeptides also can be utilized or expressed asprotein fusions. For example, an enzyme fusion can be generated to addadditional functionality to an enzyme. Examples of enzyme fusionproteins include, but are not limited to, fusions of a signal sequence,a tag such as for localization, e.g., a his₆ tag or a myc tag, or a tagfor purification, for example, a GST fusion, and a sequence fordirecting protein secretion and/or membrane association.

For example, a modified MTSP-1 polypeptide described herein is one thatis generated by expression of a nucleic acid molecule encoding theprotease domain set forth in any one of SEQ ID NOs: 1-4, 11-13 and 21-59or a sequence of amino acids that exhibits at least 65%, 70%, 75%, 80%,84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to a sequence set forth in any of SEQ ID NOs: 1-4, 11-13 and21-59.

For long-term, high-yield production of recombinant proteins, stableexpression is desired. For example, cell lines that stably express amodified MTSP-1 polypeptide can be transformed using expression vectorsthat contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells can be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells that successfully express theintroduced sequences. Resistant cells of stably transformed cells can beproliferated using tissue culture techniques appropriate to the celltypes.

Any number of selection systems can be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., (1977) Cell 11:223-232) and adeninephosphoribosyltransferase (Lowy I et al. (1980) Cell 22:817-23) genes,which can be employed in TK- or APRT-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection. For example, DHFR, which confers resistance tomethotrexate (Wigler M et al. (1980) Proc. Natl. Acad. Sci, 77:3567-70);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin F et al. (1981) J. Mol. Biol., 150:1-14); and als orpat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively, can be used. Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan or hisD, which allows cells toutilize histinol in place of histidine (Hartman S C and R C Mulligan(1988) Proc. Natl. Acad. Sci, 85:8047-8051). Visible markers, such asbut not limited to, anthocyanins, beta glucuronidase and its substrate,GUS, and luciferase and its substrate luciferin, also can be used toidentify transformants and also to quantify the amount of transient orstable protein expression attributable to a particular vector system(Rhodes C A et al. (1995) Methods Mol. Biol. 55:121-131).

The presence and expression of MTSP-1 polypeptides can be monitored. Forexample, detection of a functional polypeptide can be determined bytesting the conditioned media for hyaluronidase enzyme activity underappropriate conditions. Exemplary assays to assess the solubility andactivity of expressed proteins are provided herein.

a. Prokaryotic Cells

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins. Transformation of E. coli is a simple and rapidtechnique well known to those of skill in the art. Expression vectorsfor E. coli can contain inducible promoters, such promoters are usefulfor inducing high levels of protein expression and for expressingproteins that exhibit some toxicity to the host cells. Examples ofinducible promoters include the lac promoter, the trp promoter, thehybrid tac promoter, the T7 and SP6 RNA promoters and the temperatureregulated λPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. The cytoplasm is a reducingenvironment and for some molecules, this can result in the formation ofinsoluble inclusion bodies. Reducing agents such as dithiothreotol andβ-mercaptoethanol and denaturants, such as guanidine-HCl and urea can beused to resolubilize the proteins. An alternative approach is theexpression of proteins in the periplasmic space of bacteria whichprovides an oxidizing environment and chaperonin-like and disulfideisomerases and can lead to the production of soluble protein. Typically,a leader sequence is fused to the protein to be expressed which directsthe protein to the periplasm. The leader is then removed by signalpeptidases inside the periplasm. Examples of periplasmic-targetingleader sequences include the pelB leader from the pectate lyase gene andthe leader derived from the alkaline phosphatase gene. In some cases,periplasmic expression allows leakage of the expressed protein into theculture medium. The secretion of proteins allows quick and simplepurification from the culture supernatant. Proteins that are notsecreted can be obtained from the periplasm by osmotic lysis. Similar tocytoplasmic expression, in some cases proteins can become insoluble anddenaturants and reducing agents can be used to facilitate solubilizationand refolding. Temperature of induction and growth also can influenceexpression levels and solubility, typically temperatures between 25° C.and 37° C. are used. Typically, bacteria produce aglycosylated proteins.Thus, if proteins require glycosylation for function, glycosylation canbe added in vitro after purification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia hpolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used for production ofproteins, such as any described herein. Yeast can be transformed withepisomal replicating vectors or by stable chromosomal integration byhomologous recombination. Typically, inducible promoters are used toregulate gene expression. Examples of such promoters include GAL1, GALTand GALS and metallothionein promoters, such as CUP1, AOX1 or otherPichia or other yeast promoters. Expression vectors often include aselectable marker such as LEU2, TRP1, HIS3 and URA3 for selection andmaintenance of the transformed DNA. Proteins expressed in yeast areoften soluble. Co-expression with chaperonins such as BiP and proteindisulfide isomerase can improve expression levels and solubility.Additionally, proteins expressed in yeast can be directed for secretionusing secretion signal peptide fusions such as the yeast mating typealpha-factor secretion signal from Saccharomyces cerevisae and fusionswith yeast cell surface proteins such as the Aga2p mating adhesionreceptor or the Arxula adeninivorans glucoamylase. A protease cleavagesite such as for the Kex-2 protease, can be engineered to remove thefused sequences from the expressed polypeptides as they exit thesecretion pathway. Yeast also is capable of glycosylation atAsn-X-Ser/Thr motifs.

c. Insects and Insect Cells

Insect cells, particularly using baculovirus expression, are useful forexpressing polypeptides such as MTSP-1 polypeptides. Insect cellsexpress high levels of protein and are capable of most of thepost-translational modifications used by higher eukaryotes. Baculovirushave a restrictive host range which improves the safety and reducesregulatory concerns of eukaryotic expression. Typical expression vectorsuse a promoter for high level expression such as the polyhedrin promoterof baculovirus. Commonly used baculovirus systems include thebaculoviruses such as Autographa californica nuclear polyhedrosis virus(AcNPV), and the Bombyx mori nuclear polyhedrosis virus (BmNPV) and aninsect cell line such as Sf9 derived from Spodoptera frupperda,Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-levelexpression, the nucleotide sequence of the molecule to be expressed isfused immediately downstream of the polyhedrin initiation codon of thevirus. Mammalian secretion signals are accurately processed in insectcells and can be used to secrete the expressed protein into the culturemedium. In addition, the cell lines Pseudaletia unipuncta (A7S) andDanaus plexippus (DpN1) produce proteins with glycosylation patternssimilar to mammalian cell systems. Exemplary insect cells are those thathave been altered to reduce immunogenicity, including those with“mammalianized” baculovirus expression vectors and those lacking theenzyme FT3.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

d. Mammalian Expression

Mammalian expression systems can be used to express proteins includingMTSP-1 polypeptides. Expression constructs can be transferred tomammalian cells by viral infection such as adenovirus or by direct DNAtransfer such as liposomes, calcium phosphate, DEAE-dextran and byphysical means such as electroporation and microinjection. Expressionvectors for mammalian cells typically include an mRNA cap site, a TATAbox, a translational initiation sequence (Kozak consensus sequence) andpolyadenylation elements. IRES elements also can be added to permitbicistronic expression with another gene, such as a selectable marker.Such vectors often include transcriptional promoter-enhancers forhigh-level expression, for example the SV40 promoter-enhancer, the humancytomegalovirus (CMV) promoter and the long terminal repeat of Roussarcoma virus (RSV). These promoter-enhancers are active in many celltypes. Tissue and cell-type promoters and enhancer regions also can beused for expression. Exemplary promoter/enhancer regions include, butare not limited to, those from genes such as elastase I, insulin,immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein,alpha 1 antitrypsin, beta globin, myelin basic protein, myosin lightchain 2, and gonadotropic releasing hormone gene control. Selectablemarkers can be used to select for and maintain cells with the expressionconstruct. Examples of selectable marker genes include, but are notlimited to, hygromycin B phosphotransferase, adenosine deaminase,xanthine-guanine phosphoribosyl transferase, aminoglycosidephosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase.For example, expression can be performed in the presence of methotrexateto select for only those cells expressing the DHFR gene. Fusion withcell surface signaling molecules such as TCR-ζ and Fc_(ε)RI-γ can directexpression of the proteins in an active state on the cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude but are not limited to CHO, Balb/3T3, HeLa, MT2, mouse NSO(nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are availableadapted to serum-free media which facilitates purification of secretedproteins from the cell culture media. Examples include CHO-S cells(Invitrogen, Carlsbad, CA, Catalog number 11619-012) and the serum freeEBNA-1 cell line (Pham et al. (2003) Biotechnol. Bioeng. 84:332-42.).Cell lines also are available that are adapted to grow in specialmediums optimized for maximal expression. For example, DG44 CHO cellsare adapted to grow in suspension culture in a chemically defined,animal product-free medium.

e. Plants

Transgenic plant cells and plants can be used to express proteins suchas any described herein. Expression constructs are typically transferredto plants using direct DNA transfer such as microprojectile bombardmentand PEG-mediated transfer into protoplasts, and withagrobacterium-mediated transformation. Expression vectors can includepromoter and enhancer sequences, transcriptional termination elementsand translational control elements. Expression vectors andtransformation techniques are usually divided between dicot hosts, suchas Arabidopsis and tobacco, and monocot hosts, such as corn and rice.Examples of plant promoters used for expression include the cauliflowermosaic virus promoter, the nopaline synthase promoter, the ribosebisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.Selectable markers such as hygromycin, phosphomannose isomerase andneomycin phosphotransferase are often used to facilitate selection andmaintenance of transformed cells. Transformed plant cells can bemaintained in culture as cells, aggregates (callus tissue) orregenerated into whole plants. Transgenic plant cells also can includealgae engineered to produce hyaluronidase polypeptides. Because plantshave different glycosylation patterns than mammalian cells, this caninfluence the choice of protein produced in these hosts.

5. Purification

Host cells transformed with a nucleic acid sequence encoding a modifiedMTSP-1 polypeptide can be cultured under conditions suitable for theexpression and recovery of the encoded protein from cell culture. Theprotein produced by a recombinant cell generally is designed so that itis secreted, but it can be contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing nucleic acid encoding MTSP-1can be designed with signal sequences that facilitate direct secretionof MTSP-1 through prokaryotic or eukaryotic cell membrane.

Thus, methods for purification of polypeptides from host cells depend onthe chosen host cells and expression systems. For secreted molecules,proteins are generally purified from the culture media after removingthe cells. For intracellular expression, cells can be lysed and theproteins purified from the extract. When transgenic organisms such astransgenic plants and animals are used for expression, tissues or organscan be used as starting material to make a lysed cell extract.Additionally, transgenic animal production can include the production ofpolypeptides in milk or eggs, which can be collected, and if necessary,the proteins can be extracted and further purified using standardmethods in the art.

Proteins, such as modified MTSP-1 polypeptides, can be purified usingstandard protein purification techniques known in the art including butnot limited to, SDS-PAGE, size fractionation and size exclusionchromatography, ammonium sulfate precipitation and ionic exchangechromatography, such as anion exchange. Affinity purification techniquesalso can be utilized to improve the efficiency and purity of thepreparations. For example, antibodies, receptors and other moleculesthat bind MTSP-1 proteins can be used in affinity purification.

Expression constructs also can be engineered to add an affinity tag to aprotein such as a Small Ubiquitin-like Modifier (SUMO) tag, myc epitope,GST fusion or His6 and affinity purified with SUMO or myc antibody,glutathione resin and Ni-resin, respectively. Such tags can be joined tothe nucleotide sequence encoding a MTSP-1 as described elsewhere herein,which can facilitate purification of soluble proteins. For example, amodified MTSP-1 polypeptide can be expressed as a recombinant proteinwith one or more additional polypeptide domains added to facilitateprotein purification. Such purification facilitating domains include,but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase (Invitrogen,San Diego, CA) between the purification domain and the expressed MTSP-1polypeptide is useful to facilitate purification. One such expressionvector provides for expression of a fusion protein containing a MTSP-1polypeptide and an enterokinase cleavage site. The Small Ubiquitin-likeModifier (SUMO) tag facilitates purification on IMIAC (immobilized metalion affinity chromatography), while the enterokinase cleavage siteprovides a means for purifying the polypeptide from the fusion protein.

Purity can be assessed by any method known in the art including gelelectrophoresis, orthogonal HPLC methods, staining andspectrophotometric techniques. The expressed and purified protein can beanalyzed using any assay or method known to one of skill in the art, forexample, any described in Section 5. These include assays based on thephysical and/or functional properties of the protein, including, but notlimited to, analysis by gel electrophoresis, immunoassay and assays ofMTSP-1 activity.

6. Additional Modifications

The modified MTSP-1 polypeptides provided herein can be modified toimprove or alter pharmacokinetic and pharmacological properties. Inparticular, the modified MTSP-1 polypeptides can be conjugated to apolymer, such as a PEG moiety or dextran or sialylation to reduceimmunogenicity and/or increase half-life in serum and other body fluidsincluding vitreous humor.

a. PEGylation

Polyethylene glycol (PEG) is used in biomaterials, biotechnology andmedicine primarily because PEG is a biocompatible, nontoxic,water-soluble polymer that is typically nonimmunogenic (Zhao and Harris,ACS Symposium Series 680: 458-72, 1997). In the area of drug delivery,PEG derivatives have been widely used in covalent attachment (i.e.,“PEGylation”) to proteins to reduce immunogenicity, proteolysis andkidney clearance to increase serum half-life and to enhance solubility(Zalipsky (1995) Adv. Drug Del. Rev. 16:157-82). Similarly, PEG has beenattached to low molecular weight, relatively hydrophobic drugs toenhance solubility, reduce toxicity and alter biodistribution.Typically, PEGylated drugs are injected as solutions.

A related application is synthesis of crosslinked degradable PEGnetworks or formulations for use in drug delivery since much of the samechemistry used in design of degradable, soluble drug carriers also canbe used in design of degradable gels (Sawhney et al. (1993)Macromolecules 26: 581-87). It also is known that intermacromolecularcomplexes can be formed by mixing solutions of two complementarypolymers. Such complexes are generally stabilized by electrostaticinteractions (polyanion-polycation) and/or hydrogen bonds(polyacid-polybase) between the polymers involved, and/or by hydrophobicinteractions between the polymers in an aqueous surrounding (Krupers etal. (1996) Eur. Polym J. 32:785-790). For example, mixing solutions ofpolyacrylic acid (PAAc) and polyethylene oxide (PEO) under the properconditions results in the formation of complexes based mostly onhydrogen bonding. Dissociation of these complexes at physiologicconditions has been used for delivery of free drugs (i.e.,non-PEGylated). In addition, complexes of complementary polymers havebeen formed from homopolymers and copolymers.

Numerous reagents for PEGylation are known as are PEG moieties fortherapeutic proteins. Reagents and PEG moieties are commerciallyavailable. Such reagents include, but are not limited to, reaction ofthe polypeptide with N-hydroxysuccinimidyl (NETS) activated PEG,succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidylalpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidylbutanoate, mPEG carboxymethyl 3-hydroxybutanoic acid succinimidyl ester,homobifunctional PEG-succinimidyl propionate, homobifunctional PEGpropionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEGhydrazide, p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate,propionaldehyde PEG, mPEG butryaldehyde, branched mPEG2 butyraldehyde,mPEG acetyl, mPEG piperidone, mPEG methylketone, mPEG “linkerless”maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester,mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfonePEG-NHS, acrylate PEG-NHS, fluorescein PEG-NHS, and biotin PEG-NETS (seee.g., Monfardini et al. (1995) Bioconjugate Chem. 6:62-69; Veronese etal. (1997) J. Bioactive Compatible Polymers 12:196-207; U.S. Pat. Nos.5,672,662; 5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337;5,122,614; 5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581;5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237;6,113,906; 6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339;6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S.2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430;U.S. 2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S. 2004/0013637;US 2004/0235734; WO 05/00360; U.S. 2005/0114037; U.S. 2005/0171328; U.S.2005/0209416; EP 01064951; EP 0822199; WO 00176640; WO 00/02017; WO02/49673; WO 94/28024; and WO 01/87925).

In one example, the polyethylene glycol has a molecular weight rangingfrom about 3 kD to about 50 kD, and typically from about 5 kD to about30 kD. Covalent attachment of the PEG to the drug (known as“PEGylation”) can be accomplished by known chemical synthesistechniques. For example, the PEGylation of protein can be accomplishedby reacting NETS-activated PEG with the protein under suitable reactionconditions.

While numerous reactions have been described for PEGylation, those thatare most generally applicable confer directionality, use mild reactionconditions, and do not necessitate extensive downstream processing toremove toxic catalysts or bi-products. For instance, monomethoxy PEG(mPEG) has only one reactive terminal hydroxyl, and thus its use limitssome of the heterogeneity of the resulting PEG-protein product mixture.Activation of the hydroxyl group at the end of the polymer opposite tothe terminal methoxy group is generally necessary to accomplishefficient protein PEGylation, with the aim being to make the derivatizedPEG more susceptible to nucleophilic attack. The attacking nucleophileis usually the epsilon-amino group of a lysyl residue, but other aminesalso can react (e.g., the N-terminal alpha-amine or the ring amines ofhistidine) if local conditions are favorable. A more directed attachmentis possible in proteins containing a single lysine or cysteine. Thelatter residue can be targeted by PEG-maleimide for thiol-specificmodification. Alternatively, PEG hydrazide can be reacted with aperiodate oxidized hyaluronan-degrading enzyme and reduced in thepresence of NaCNBH₃. More specifically, PEGylated CMP sugars can bereacted with a hyaluronan-degrading enzyme in the presence ofappropriate glycosyl-transferases. One technique is the “PEGylation”technique where a number of polymeric molecules are coupled to thepolypeptide in question. When using this technique the immune system hasdifficulties in recognizing the epitopes on the polypeptide's surfaceresponsible for the formation of antibodies, thereby reducing the immuneresponse. For polypeptides introduced directly into the circulatorysystem of the human body to give a particular physiological effect(i.e., pharmaceuticals) the typical potential immune response is an IgGand/or IgM response, while polypeptides which are inhaled through therespiratory system (i.e., industrial polypeptide) potentially can causean IgE response (i.e., allergic response). One of the theoriesexplaining the reduced immune response is that the polymeric molecule(s)shield(s) epitope(s) on the surface of the polypeptide responsible forthe immune response leading to antibody formation. Another theory or atleast a partial factor is that the heavier the conjugate is, the morereduced immune response is obtained.

Typically, to make the PEGylated modified MTSP-1 polypeptide providedherein, PEG moieties are conjugated, via covalent attachment, to thepolypeptides. Techniques for PEGylation include, but are not limited to,specialized linkers and coupling chemistries (see e.g., Roberts et al.,(2002) Adv. Drug Deliv. Rev. 54:459-476), attachment of multiple PEGmoieties to a single conjugation site (such as via use of branched PEGs;see e.g., Guiotto et al. (2002) Bioorg. Med. Chem. Lett. 12:177-180),site-specific PEGylation and/or mono-PEGylation (see e.g., Chapman etal. (1999) Nature Biotech. 17:780-783), and site-directed enzymaticPEGylation (see e.g., Sato (2002), Adv. Drug Deliv. Rev., 54:487-504).Methods and techniques described in the art can produce proteins having1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 PEG or PEG derivativesattached to a single protein molecule (see e.g., U.S. Patent PublicationNo. 2006/0104968).

b. Fusion Proteins

Preparation of fusions of therapeutic proteins to other moieties, suchas PEGylation, conjugation to albumin, targeting moieties, such asantibodies and antigen binding fragments thereof, immunoglobulins, fcfusions, fusion with albumin (HSA), XTEN fusion proteins, modificationof glycosylation patterns are known (see, Strohl (2015) BioDrugs29:215-239 for a review of a variety of fusion proteins for improvingpharmacokinetic properties of therapeutic proteins). Any of these knownmodalities for improving pharmacological properties of therapeutics canbe applied to the modified MTSP-1 polypeptides provided herein.

Fusion proteins containing a modified MTSP-1 polypeptide provided hereinand one or more other polypeptides also are provided. Pharmaceuticalcompositions containing such fusion proteins formulated foradministration by a suitable route are provided. Fusion proteins areformed by linking in any order the modified MTSP-1 polypeptide andanother polypeptide, such as an antibody or fragment thereof, growthfactor, receptor, ligand and other such agent for the purposes offacilitating the purification of a protease, altering thepharmacodynamic properties of a MTSP-1 polypeptide by directing themodified MTSP-1 polypeptide to a targeted cell or tissue, and/orincreasing the expression or secretion of a modified MTSP-1 polypeptide.Within a modified MTSP-1 polypeptide fusion protein, the modified MTSP-1polypeptide can correspond to all or a catalytically active portionthereof of a MTSP-1 polypeptide. In some embodiments, the MTSP-1polypeptide or catalytically active portion thereof is a modified MTSP-1polypeptide provided herein. Fusion proteins provided herein retainsubstantially all of their specificity and/or selectivity for complementprotein C3. Generally, MTSP-1 fusion polypeptides retain at least about30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% substrate specificityand/or selectivity compared with a non-fusion MTSP-1 polypeptide,including 96%, 97%, 98%, 99% or greater substrate specificity comparedwith a non-fusion MTSP-1 polypeptide.

Linkage of a modified MTSP-1 polypeptide and another polypeptide can beeffected directly or indirectly via a linker. In one example, linkagecan be by chemical linkage, such as via heterobifunctional agents orthiol linkages or other such linkages. Fusion of a MTSP-1 polypeptide toanother polypeptide can be to the N- or C-terminus of the MTSP-1polypeptide. Non-limiting examples of polypeptides that can be used infusion proteins with a modified MTSP-1 polypeptide provided hereininclude, for example, a GST (glutathione S-transferase) polypeptide, Fcdomain from immunoglobulin G, or a heterologous signal sequence. Thefusion proteins can contain additional components, such as E. colimaltose binding protein (MBP) that aid in uptake of the protein by cells(see, International Patent Publication No. WO 01/32711).

A MTSP-1 polypeptide fusion protein can be produced by standardrecombinant techniques. For example, DNA fragments encoding thedifferent polypeptide sequences can be ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley & Sons, 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).A MTSP-1-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the MTSP-1polypeptide.

Fc fusion proteins, fusion to human serum albumin, fusion tocarboxy-terminal peptide are known modifications for improvingpharmacokinetics of peptide or biologic drugs. Among these isconjugation to either linear or branched-chain monomethoxy poly-ethyleneglycol (PEG), resulting in increases in the molecular mass andhydrodynamic radius, and a decrease in the rate of glomerular filtrationby the kidney.

Another approach to for improving pharmacokinetic parameters includesmodification of glycosylation patterns, resulting in reduced clearanceand extension of half-life.

7. Nucleic Acid Molecules

Nucleic acid molecules encoding MTSP-1 polypeptides are provided herein.Nucleic acid molecules include allelic variants or splice variants ofany encoded MTSP-1 polypeptide, or catalytically active portion thereof.In one embodiment, nucleic acid molecules provided herein have at least50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, or 99% sequenceidentity or hybridize under conditions of medium or high stringencyalong at least 70% of the full-length of any nucleic acid encoded MTSP-1polypeptide, or catalytically active portion thereof. In anotherembodiment, a nucleic acid molecule can include those with degeneratecodon sequences of any of the MTSP-1 polypeptides or catalyticallyactive portions thereof such as those provided herein. Exemplary nucleicacid molecules can encode scaffold or modified proteases, orcatalytically active portions thereof.

Nucleic acid molecules, or fusion proteins containing a catalyticallyactive portion of a nucleic acid molecule, operably-linked to apromoter, such as an inducible promoter for expression in mammaliancells also are provided. Such promoters include, but are not limited to,CMV and SV40 promoters; adenovirus promoters, such as the E2 genepromoter, which is responsive to the HPV E7 oncoprotein; a PV promoter,such as the PBV p89 promoter that is responsive to the PV E2 protein;and other promoters that are activated by the HIV or PV or oncogenes.

A MTSP-1 protease provided herein, also can be delivered to the cells ingene transfer vectors. The transfer vectors also can encode additionalother therapeutic agent(s) for treatment of the disease or disorder,such as Rheumatoid Arthritis or cardiovascular disease or AMD or DGF,for which the protease is administered. Transfer vectors encoding aprotease can be used systemically, by administering the nucleic acid toa subject. For example, the transfer vector can be a viral vector, suchas an adenovirus vector. Vectors encoding a protease also can beincorporated into stem cells and such stem cells administered to asubject such as by transplanting or engrafting the stem cells at sitesfor therapy. For example, mesenchymal stem cells (MSCs) can beengineered to express a protease and such MSCs engrafted at a transplantsite for therapy.

G. COMPOSITIONS, FORMULATIONS AND DOSAGES

Pharmaceutical compositions containing modified MTSP-1 polypeptides,modified MTSP-1 fusion proteins or encoding nucleic acid molecules, canbe formulated in any conventional manner by mixing a selected amount ofthe polypeptide with one or more physiologically acceptable carriers orexcipients. Selection of the carrier or excipient is within the skill ofthe administering professional and can depend upon a number ofparameters. These include, for example, the mode of administration(i.e., systemic, oral, nasal, pulmonary, local, topical or any othermode) and disorder treated. The pharmaceutical compositions providedherein can be formulated for single dosage (direct) administration orfor dilution or other modification. The concentrations of the compoundsin the formulations are effective for delivery of an amount, uponadministration, that is effective for the intended treatment. Typically,the compositions are formulated for single dosage administration. Toformulate a composition, the weight fraction of a compound or mixturethereof is dissolved, suspended, dispersed or otherwise mixed in aselected vehicle at an effective concentration such that the treatedcondition is relieved or ameliorated. Pharmaceutical carriers orvehicles suitable for administration of the compounds provided hereininclude any such carriers known to those skilled in the art to besuitable for the particular mode of administration.

1. Administration of Modified MTSP-1 Polypeptides

The polypeptides can be formulated as the sole pharmaceutically activeingredient in the composition or can be combined with other activeingredients. The polypeptides can be targeted for delivery, such as byconjugation to a targeting agent, such as an antibody. Liposomalsuspensions, including tissue-targeted liposomes, also can be suitableas pharmaceutically acceptable carriers. These can be prepared accordingto methods known to those skilled in the art. For example, liposomeformulations can be prepared as described in U.S. Pat. No. 4,522,811.Liposomal delivery also can include slow release formulations, includingpharmaceutical matrices such as collagen gels and liposomes modifiedwith fibronectin (see, for example, Weiner et al. (1985) J Pharm Sci.74(9): 922-5).

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the subject treated. Thetherapeutically effective concentration can be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein.

The MTSP-1 polypeptides provided herein (i.e., active compounds) can beadministered in vitro, ex vivo, or in vivo by contacting a mixture, suchas a body fluid, such as the vitreous, or other tissue sample, with aMTSP-1 polypeptide provided herein, including any of the modified MTSP-1polypeptides provided herein. For example, when administering a compoundex vivo, a body fluid or tissue sample from a subject can be contactedwith the MTSP-1 polypeptides that are coated on a tube or filter, suchas for example, a true or filter in a bypass machine. When administeringin vivo, the active compounds can be administered by any appropriateroute, for example, orally, nasally, pulmonary, parenterally,intravenously, intradermally, intravitreally, periocularly,subcutaneously, or topically, in liquid, semi-liquid or solid form andare formulated in a manner suitable for each route of administration.Determination of dosage is within the skill of the physician, and can bea function of the particular disorder, route of administration andsubject. Exemplary dosages will be dosed at 0.1-1 mg.

The modified MTSP-1 polypeptide and physiologically acceptable salts andsolvates can be formulated for administration by inhalation (eitherthrough the mouth or the nose), oral, transdermal, pulmonary, parenteralor rectal administration. For administration by inhalation, the modifiedMTSP-1 polypeptide can be delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator, can be formulated containing a powder mix of a therapeuticcompound and a suitable powder base such as lactose or starch.

For pulmonary administration to the lungs, the modified MTSP-1polypeptide can be delivered in the form of an aerosol spraypresentation from a nebulizer, turbonebulizer, ormicroprocessor-controlled metered dose oral inhaler with the use of asuitable propellant. Generally, particle size of the aerosol is small,such as in the range of 0.5 to 5 microns. In the case of apharmaceutical composition formulated for pulmonary administration,detergent surfactants are not typically used. Pulmonary drug delivery isa promising non-invasive method of systemic administration. The lungsrepresent an attractive route for drug delivery, mainly due to the highsurface area for absorption, thin alveolar epithelium, extensivevascularization, lack of hepatic first-pass metabolism, and relativelylow metabolic activity.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets, pills, liquid suspensions, or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets can be coated by methodswell known in the art. Liquid preparations for oral administration cantake the form of, for example, solutions, syrups or suspensions, or theycan be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations also cancontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

Preparations for oral administration can be formulated for controlledrelease of the active compound. For buccal administration thecompositions can take the form of tablets or lozenges formulated inconventional manner.

The modified MTSP-1 polypeptides can be formulated as a depotpreparation. Such long-acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the therapeutic compoundscan be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The modified MTSP-1 polypeptide can be formulated for parenteraladministration by injection (e.g., by bolus injection or continuousinfusion). Formulations for injection can be presented in unit dosageform (e.g., in ampoules or in multi-dose containers) with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be inpowder-lyophilized form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The modified MTSP-1 polypeptides can be formulated for ocular orophthalmic delivery. Ocular drug delivery includes, for example,topical, oral or systemic, and/or injected. For example, a modifiedMTSP-1 polypeptide(s) or pharmaceutical composition containing amodified MTSP-1 polypeptide(s) can be administered topically, such as inthe form of eye drops. In another example, a modified MTSP-1polypeptide(s) or pharmaceutical composition containing a modifiedMTSP-1 polypeptide(s) can be administered by periocular and/orintravitreal administration, such as, for example, by periocular orintravitreal injection(s).

The modified MTSP-1 polypeptides or pharmaceutical compositioncontaining modified MTSP-1 polypeptides or nucleic acids encodingmodified MTSP-1 polypeptides can be formulated for systemicadministration for treatment of DGF. In another example, the modifiedMTSP-1 polypeptides or pharmaceutical composition containing modifiedMTSP-1 polypeptides or nucleic acids encoding modified MTSP-1polypeptides are directly infused or injected into the kidney or intothe tissues or organs adjacent or surrounding the transplanted kidney.The modified MTSP-1 polypeptides or pharmaceutical compositioncontaining modified MTSP-1 polypeptides can be administered before thetime of allograft transplantation or at the time of transplantation withadministration continuing in a chronic fashion, and/or can beadministered during a rejection episode in the event such an episodedoes occur.

The pharmaceutical compositions can be formulated for local or topicalapplication, such as for topical application to the skin (transdermal)and mucous membranes, such as in the eye, in the form of gels, creams,and lotions and for application to the eye or for intracisternal orintraspinal application. Such solutions, particularly those intended forophthalmic use, can be formulated as 0.01%-10% isotonic solutions and pHabout 5-7 with appropriate salts. The compounds can be formulated asaerosols for topical application, such as by inhalation (see, forexample, U.S. Pat. Nos. 4,044,126, 4,414,209 and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatmentinflammatory diseases, particularly asthma).

The concentration of active compound in the drug composition depends onabsorption, inactivation and excretion rates of the active compound, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art. As described further herein, dosages canbe determined empirically using comparisons of properties and activities(e.g., cleavage of one or more complement proteins) of the modifiedMTSP-1 polypeptide compared to the unmodified and/or wild type and/orreference MTSP-1 polypeptide.

The compositions, if desired, can be presented in a package, in a kit ordispenser device, that can contain one or more unit dosage formscontaining the active ingredient. In some examples, the composition canbe coated on a device, such as for example on a tube or filter in, forexample, a bypass machine. The package, for example, contains metal orplastic foil, such as a blister pack. The pack or dispenser device canbe accompanied by instructions for administration. The compositionscontaining the active agents can be packaged as articles of manufacturecontaining packaging material, an agent provided herein, and a labelthat indicates the disorder for which the agent is provided.

Also provided are compositions containing nucleic acid molecules,including expression vectors, encoding the MTSP-1 polypeptides. In someembodiments, the compositions of nucleic acid molecules encoding theMTSP-1 polypeptides and expression vectors encoding them are suitablefor gene therapy. Rather than deliver the protein, nucleic acid can beadministered in vivo, such as systemically or by other route, or exvivo, such as by removal of cells, including lymphocytes, introductionof the nucleic acid therein, and reintroduction into the host or acompatible recipient.

2. Administration of Nucleic Acids Encoding Modified MTSP-1 Polypeptides(Gene Therapy)

MTSP-1 polypeptides can be delivered to cells and tissues by expressionof nucleic acid molecules. MTSP-1 polypeptides can be administered asnucleic acid molecules encoding MTSP-1 polypeptides, including ex vivotechniques and direct in vivo expression. Nucleic acids can be deliveredto cells and tissues by any method known to those of skill in the art.The isolated nucleic acid can be incorporated into vectors for furthermanipulation. Exemplary nucleic acids are any that encode or thathybridize under medium to high stringency to a nucleic acid that encodesa MTSP-1 polypeptide, or catalytically active portion thereof having asequence of amino acids set forth in any of SEQ ID NOS: 21-59. Exemplarynucleic acid molecules can encode modified MTSP-1 polypeptides, orcatalytically active portions thereof.

Methods for administering MTSP-1 polypeptides by expression of encodingnucleic acid molecules include administration of recombinant vectors.The vector can be designed to remain episomal, such as by inclusion ofan origin of replication or can be designed to integrate into achromosome in the cell. MTSP-1 polypeptides also can be used in ex vivogene expression therapy using vectors. Suitable gene therapy vectors andmethods of delivery are known to those of skill in the art. For example,cells can be engineered to express a modified MTSP-1 polypeptide, suchas by integrating MTSP-1 polypeptide encoding nucleic acid into agenomic location, either operatively linked to regulatory sequences orsuch that it is placed operatively linked to regulatory sequences in agenomic location. Such cells then can be administered locally orsystemically to a subject, such as a patient in need of treatment.Exemplary vectors for in vivo and ex vivo gene therapy include viralvectors, and non-viral vectors such as for example, liposomes orartificial chromosomes.

Viral vectors, including, for example adenoviruses, herpes viruses,adeno-associated viruses (AAV), retroviruses, such as lentiviruses, EBV,SV40, cytomegalovirus vectors, vaccinia virus vectors, and othersdesigned for gene therapy can be employed. The vectors can be those thatremain episomal or those that can integrate into chromosomes of thetreated subject. A modified MTSP-1 polypeptide can be expressed by avirus, which is administered to a subject in need of treatment. Virusvectors suitable for gene therapy include adenovirus, adeno-associatedvirus, retroviruses, lentiviruses and others noted above. For example,adenovirus expression technology is well-known in the art and adenovirusproduction and administration methods also are well known. Adenovirusserotypes are available, for example, from the American Type CultureCollection (ATCC, Rockville, MD). Adenovirus can be used ex vivo, forexample, cells are isolated from a patient in need of treatment, andtransduced with a modified MTSP-1 polypeptide-expressing adenovirusvector. After a suitable culturing period, the transduced cells areadministered to a subject, locally and/or systemically. Alternatively,MTSP-1 polypeptide-expressing adenovirus particles are isolated andformulated in a pharmaceutically-acceptable carrier for delivery of atherapeutically effective amount to prevent, treat or ameliorate adisease or condition of a subject. In one embodiment, the disease to betreated is caused by complement activation. Typically, adenovirusparticles are delivered at a dose ranging from 1 particle to 10¹⁴particles per kilogram subject weight, generally between 10⁶ or 10⁸particles to 10¹² particles per kilogram subject weight.

The nucleic acid molecules can be introduced into artificial chromosomesand other non-viral vectors. Artificial chromosomes, such as ACES (see,Lindenbaum et al. (2004) Nucleic Acids Res. 32(21):e172) can beengineered to encode and express the MTSP-1 polypeptide. Briefly,mammalian artificial chromosomes (MACs) provide a means to introducelarge payloads of genetic information into the cell in an autonomouslyreplicating, non-integrating format. Unique among MACs, the mammaliansatellite DNA-based Artificial Chromosome Expression System (ACES) canbe reproducibly generated de novo in cell lines of different species andreadily purified from the host cells' chromosomes. Purified mammalianACES can then be re-introduced into a variety of recipient cell lineswhere they have been stably maintained for extended periods in theabsence of selective pressure using an ACE System. Using this approach,specific loading of one or two gene targets has been achieved in LMTK(−)and CHO cells.

Another method for introducing nucleic acids encoding the modifiedMTSP-1 polypeptides is a two-step gene replacement technique in yeast,starting with a complete adenovirus genome (Ad2; Ketner et al. (1994)Proc. Natl. Acad. Sci. USA 91: 6186-6190) cloned in a Yeast ArtificialChromosome (YAC) and a plasmid containing adenovirus sequences to targeta specific region in the YAC clone, an expression cassette for the geneof interest and a positive and negative selectable marker. YACs are ofparticular interest because they permit incorporation of larger genes.This approach can be used for construction of adenovirus-based vectorsbearing nucleic acids encoding any of the described modified MTSP-1polypeptides for gene transfer to mammalian cells or whole animals.

The nucleic acids can be encapsulated in a vehicle, such as a liposome,or introduced into a cells, such as a bacterial cell, particularly anattenuated bacterium or introduced into a viral vector. For example,when liposomes are employed, proteins that bind to a cell surfacemembrane protein associated with endocytosis can be used for targetingand/or to facilitate uptake, e.g., capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life.

In some embodiments, it is desirable to provide a nucleic acid sourcewith an agent that targets cells, such as an antibody specific for acell surface membrane protein or a target cell, or a ligand for areceptor on a target cell. Polynucleotides and expression vectorsprovided herein can be made by any suitable method. Further provided arenucleic acid vectors containing nucleic acid molecules as describedabove. Further provided are nucleic acid vectors containing nucleic acidmolecules as described above and cells containing these vectors.

For ex vivo and in vivo methods, nucleic acid molecules encoding theMTSP-1 polypeptide are introduced into cells that are from a suitabledonor or the subject to be treated. Cells into which a nucleic acid canbe introduced for purposes of therapy include, for example, any desired,available cell type appropriate for the disease or condition to betreated including, but not limited to, epithelial cells, endothelialcells, keratinocytes, fibroblasts, muscle cells, hepatocytes; bloodcells such as T lymphocytes, B lymphocytes, monocytes, macrophages,neutrophils, eosinophils, megakaryocytes, granulocytes; various stem orprogenitor cells, including hematopoietic stem or progenitor cells,e.g., such as stem cells obtained from bone marrow, umbilical cordblood, peripheral blood, fetal liver, and other sources thereof.

For ex vivo treatment, cells from a donor compatible with the subject tobe treated or cells from a subject to be treated are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the subject. Treatment includes directadministration, such as, for example, encapsulated within porousmembranes, which are implanted into the patient (see, e.g., U.S. Pat.Nos. 4,892,538 and 5,283,187). Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomesand cationic lipids (e.g., DOTMA, DOPE and DC-Chol) electroporation,microinjection, cell fusion, DEAE-dextran, and calcium phosphateprecipitation methods. Methods of DNA delivery can be used to expressMTSP-1 polypeptides in vivo. Such methods include liposome delivery ofnucleic acids and naked DNA delivery, including local and systemicdelivery such as using electroporation, ultrasound and calcium-phosphatedelivery. Other techniques include microinjection, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer andspheroplast fusion.

In vivo expression of a MTSP-1 polypeptide can be linked to expressionof additional molecules. For example, expression of a MTSP-1 polypeptidecan be linked with expression of a cytotoxic product such as in anengineered virus or expressed in a cytotoxic virus. Such viruses can betargeted to a particular cell type that is a target for a therapeuticeffect. The expressed MTSP-1 polypeptide can be used to enhance thecytotoxicity of the virus.

In vivo expression of a MTSP-1 polypeptide can include operativelylinking a MTSP-1 polypeptide encoding nucleic acid molecule to specificregulatory sequences such as a cell-specific or tissue-specificpromoter. MTSP-1 polypeptides also can be expressed from vectors thatspecifically infect and/or replicate in target cell types and/ortissues. Inducible promoters can be used to selectively regulate MTSP-1polypeptide expression.

Nucleic acid molecules, as naked nucleic acids or in vectors, artificialchromosomes, liposomes and other vehicles can be administered to thesubject by systemic administration, topical, local and other routes ofadministration. When systemic and in vivo, the nucleic acid molecule orvehicle containing the nucleic acid molecule can be targeted to a cell.

Administration also can be direct, such as by administration of a vectoror cells that typically targets a cell or tissue. For example, tumorcells and proliferating cells can be targeted cells for in vivoexpression of MTSP-1 polypeptides. Cells used for in vivo expression ofa MTSP-1 polypeptide also include cells autologous to the patient. Suchcells can be removed from a patient, nucleic acids for expression of aMTSP-1 polypeptide introduced, and then administered to a patient suchas by injection or engraftment.

H. THERAPEUTIC USES AND METHODS OF TREATMENT

The modified MTSP-1 polypeptides provided herein target complementprotein C3 and permit modulation of complement-mediated diseases anddisorders. Therapeutic proteases, such as the modified MTSP-1polypeptides provided herein, have many potential advantages overtraditional therapeutic approaches. Chief among them is the ability toinactivate disease targets in a catalytic manner (i.e. a one to manystoichiometry). Thus, proteases can maintain effective regulation atconcentrations significantly below the target concentration. Additionaldifferentiating advantages include (1) irreversible inactivation; (2)low dosing; (3) decreased dosing frequency; (4) small molecular size;(5) the ability to target post-translational modifications; (6) theability to neutralize high target concentrations; and (7) the ability totarget away from the active site. As a therapeutic, a protease muststill exhibit the following characteristics: (1) access to the moleculartarget (extracellular), and (2) possess sufficiently stringentspecificity for a target critical to a disease state. The modifiedMTSP-1 polypeptides provided herein can be used in the treatment ofcomplement-mediated diseases and disorders.

The skilled artisan understands the role of the complement system indisease processes and is aware of a variety of such diseases. Providedis a brief discussion of exemplary diseases and the role of thecomplement protein C3 in their etiology and pathology. The modifiedMTSP-1 polypeptides and nucleic acid molecules provided herein can beused for treatment of any condition for which activation of thecomplement pathway is implicated, particularly inflammatory conditionsincluding acute inflammatory conditions, such as septic shock, andchronic inflammatory conditions, such as Rheumatoid Arthritis (RA).Acute and inflammatory conditions can be manifested as animmune-mediated disease such as for example autoimmune disease or tissueinjury caused by immune-complex-mediated inflammation. Acomplement-mediated inflammatory condition also can be manifested as aneurodegenerative or cardiovascular disease that have inflammatorycomponents. This section provides exemplary uses of, and administrationmethods for, modified MTSP-1 polypeptides provided herein. Thesedescribed therapies are exemplary and do not limit the applications ofthe modified MTSP-1 polypeptides provided herein. Such methods include,but are not limited to, methods of treatment of physiological andmedical conditions described and listed below. Such methods include, butare not limited to, methods of treatment of age-related maculardegeneration (AMD), geographic atrophy (GA), paroxysmal nocturnalhemoglobinuria (PNH), renal delayed graft function (DGF), sepsis,Rheumatoid arthritis (RA), membranoproliferative glomerulonephritis(MPGN), lupus erythematosus, Multiple Sclerosis (MS), Myasthenia gravis(MG), asthma, inflammatory bowel disease, respiratory distress syndrome,immune complex (IC)-mediated acute inflammatory tissue injury,multi-organ failure, Alzheimer's Disease (AD), Ischemia-reperfusioninjuries caused by events or treatments such as myocardial infarct (MI),stroke, cardiopulmonary bypass (CPB) or coronary artery bypass graft,angioplasty, or hemodialysis, chronic obstructive pulmonary disease(COPD), idiopathic pulmonary fibrosis (IPF) and/or Guillain Barresyndrome.

Treatment of diseases and conditions with modified MTSP-1 polypeptidesprovided herein can be effected by any suitable route of administrationusing suitable formulations as described herein including, but notlimited to, subcutaneous injection, oral, intravitreal, periocular andtransdermal administration. If necessary, a particular dosage andduration and treatment protocol can be empirically determined orextrapolated. For example, exemplary doses of wild type or referenceMTSP-1 polypeptides can be used as a starting point to determineappropriate dosages. Modified MTSP-1 polypeptides that have morespecificity and/or selectivity compared to a wild type or referenceMTSP-1 polypeptide can be effective at reduced dosage amounts and orfrequencies. Dosage levels can be determined based on a variety offactors, such as body weight of the individual, general health, age, theactivity of the specific compound employed, sex, diet, time ofadministration, rate of excretion, drug combination, the severity andcourse of the disease, and the patient's disposition to the disease andthe judgment of the treating physician. The amount of active ingredientthat can be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration.

Upon improvement of a patient's condition, a maintenance dose of acompound or compositions can be administered, if necessary; and thedosage, the dosage form, or frequency of administration, or acombination thereof can be modified. In some cases, a subject canrequire intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

1. Disease Mediated by Complement Activation

The complement cascade is a dual-edged sword, causing protection againstbacterial and viral invasion by promoting phagocytosis and inflammation.Conversely, even when complement is functioning normally, it cancontribute to the development of disease by promoting local inflammationand damage to tissues. Thus, pathological effects are mediated by thesame mediators that are responsible for the protective roles ofcomplement. For example, the anaphylactic and chemotactic peptide C5adrives inflammation by recruiting and activating neutrophils, C3a cancause pathological activation of other phagocytes, and the membraneattack complex can kill or injure cells. In one example, such as in manyautoimmune diseases, complement produces tissue damage because it isactivated under inappropriate circumstances such as by antibody to hosttissues. In other situations, complement can be activated normally, suchas by septicemia, but still contributes to disease progression, such asin respiratory distress syndrome. Pathologically, complement can causesubstantial damage to blood vessels (vasculitis), kidney basementmembrane and attached endothelial and epithelial cells (nephritis),joint synovium (arthritis), and erythrocytes (hemolysis) if it is notadequately controlled.

Complement has a role in immuno-pathogenesis of a number of disorders,including autoimmune diseases such as rheumatoid arthritis (see, e.g.,Wang et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:8955-8959; Moxley etal. (1987) Arthritis & Rheumatism 30:1097-1104), lupus erythematosus(Wang et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 90:8563-8568; andBuyon et al. (1992) Arthritis Rheum. 35:1028-1037) and acuteglomerulonephritis (Couser et al. (1995) J Am Soc Nephrol. 5:1888-1894).Other pathologies that involve activation of the complement systeminclude sepsis (see, e.g., Stove et al. (1996) Clin Diag Lab Immunol3:175-183; Hack et al. (1989) Am. J. Med. 86:20-26), respiratorydistress syndrome (see, e.g., Zilow et al. (1990) Clin. Exp. Immunol.79:151-157; and Stevens et al. (1986) J. Clin. Invest. 77:1812-1816),multiorgan failure (see, e.g., Hecke et al. (1997) Shock 7:74; andHeideman et al. (1984) J. Trauma 24:1038-1043), ischemia-reperfusioninjury such as occurs in cardiovascular disease such as stroke ormyocardial infarct (Austen W G et al. (2003) Int J Immunopathol Pharm16(1):1-8), age-related macular degeneration (Bradley et al. (2011) Eye25: 683-693; Gemenetzi et al. (2016) Eye 30: 1-14) and renal delayedgraft function (Danobeitia et al. (2013) [abstract]. Am J Transplant. 13(suppl 5); Yu et al. (2016) Am J Transplant 16(9):2589-2597; Castallanoet al. (2010) Am J Pathol 176(4):1648-1659). Some exemplary examples ofcomplement-mediated diseases are described below.

a. Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory illness. It is anautoimmune disease in which the immune system attacks normal tissuecomponents as if they were invading pathogens. The inflammationassociated with rheumatoid arthritis primarily attacks the linings ofthe joints. The membranes lining the blood vessels, heart, and lungsalso can become inflamed. RA is characterized by activated B cells andplasma cells that are present in inflamed synovium, and in establisheddisease lymphoid follicles and germinal centers. This results in highlevels of local immunoglobulin production and the deposition of immunecomplexes, which can include IgG and IgM rheumatoid factors, in thesynovium and in association with articular cartilage which can serve asinitiators of the complement cascade. Elevated levels of complementcomponents, such as C3a, C5a, and C5b-9 have been found within theinflamed rheumatoid joints. These complement components can exacerbatethe inflammation associated with RA by inducing a variety ofproinflammatory activities such as for example, alterations in vascularpermeability, leukocyte chemotaxis, and the activation and lysis ofmultiple cell types.

b. Sepsis

Sepsis is a disease caused by a serious infection, such as a bacterialinfection, leading to a systemic inflammatory response. The bacterialcell wall component, lipopolysaccharide, is often associated withsepsis, although other bacterial, viral, and fungal infections canstimulate septic symptoms. Septic shock often results if the naturalimmune system of the body is unable to defend against an invadingmicroorganism such that, for example, the pro-inflammatory consequencesof the immune response is damaging to host tissues. The early stages ofsepsis are characterized by excessive complement activation resulting inincreased production of complement anaphylatoxins, such as C3a, C4a, andC5a which act to increase vascular permeability, stimulate superoxideproduction from neutrophils and stimulate histamine release. The actionsof C5a can contribute to a productive immune response to a bacterialinfection, but if left unregulated, C5a also can be severely damaging.In an E. coli-induced model of inflammation, blockade of C5a improvedthe outcome of septic animals by limiting C5a-mediated neutrophilactivation that can lead to neutrophil-mediated tissue injury.

The continued impairment of the innate immune response to a bacterialinfection often leads to chronic sepsis or septic shock, which can belife-threatening. In the late stage of sepsis, it is the “dormant”activity of neutrophils, as opposed to the hyperactivity that occurs inthe early phases, that contributes to continued disease. In the latestage, the major functions of neutrophils including chemotaxis,respiratory burst activity, and ability for bacterial killing arereduced. Complement, and in particular C5a, also plays a role in thelater stages of sepsis. Excessive production of C5a during sepsis isassociated with the “deactivation” of blood neutrophils, a process thathas been linked to C5a-induced down regulation of its own receptor,C5aR, on neutrophils (Guo et al. (2003) FASEB J 13:1889). The reducedlevels of C5aR on neutrophils correlates with a diminished ability ofblood neutrophils to bind C5a, impaired chemotactic responses, a loss ofsuperoxide productions, and impaired bactericidal activity. C5aR levels,however, can begin to “recover” at later stages of sepsis and correlatewith instances of beneficial disease outcome.

c. Multiple Sclerosis

Multiple sclerosis (MS) and its animal model experimental allergicencephalomyelitis (EAE) are inflammatory demyelinating diseases of thecentral nervous system (CNS). In MS, inflammation of nervous tissuecauses the loss of myelin, a fatty material which acts as a sort ofprotective insulation for the nerve fibers in the brain and spinal cord.This demyelination leaves multiple areas of scar tissue (sclerosis)along the covering of the nerve cells, which disrupts the ability of thenerves to conduct electrical impulses to and from the brain, producingthe various symptoms of MS. MS is mediated by activated lymphocytes,macrophages/microglia and the complement system. Complement activationcan contribute to the pathogenesis of these diseases through its dualrole: the ability of activated terminal complex C5b-9 to promotedemyelination and the capacity of sublytic C5b-9 to protectoligodendrocytes (OLG) from apoptosis.

d. Alzheimer's Disease

Alzheimer's disease (AD) is characterized by tangles (abnormal pairedhelical filaments of the protein tau, which normally binds tomicrotubules) and plaques (extracellular deposits composed primarily ofbeta-amyloid protein) within the brain. Although, the precise cause ofAD is not entirely clear, chronic neuroinflammation in affected regionsof AD brains indicate that proinflammatory mediators can play a role.The tangles and plaques within an AD brain are deposited with activatedcomplement fragments, such as for example, C4d and C3d. Likewise,dystrophic neurites in AD brain can be immunostained for MAC, indicatingautocatalytic attack of these neurites and concomitant neurite loss inAD. Activation of complement in AD occurs by an antibody-independentmechanism induced by aggregated amyloid-beta protein. Further, thecomplement cascade can be activated by the pentraxins, C-reactiveprotein (CRP), and amyloid P (AP) which are all upregulated in AD(McGeer et al., (2002) Trends Mol Med 8:519). The activation ofcomplement in AD, marked by increases in complement mediators, is notadequately controlled by a compensatory upregulation of complementregulatory proteins such as, for example, CD59. Thus, theproinflammatory consequences of complement activation exacerbates ADprogression and likely contributes to neurite destruction.

e. Ischemia-Reperfusion Injury

Ischemia-reperfusion injury is the injury sustained after an ischemicevent and subsequent restoration of blood flow and results from theinflammatory response to a hypoxic insult. Ischemia-reperfusion damagecan be acute as during cardiac surgery procedures, such as for examplefollowing open heart surgery or angioplasty, or chronic as withcongestive heart failure or occlusive cardiovascular disease. Examplesof injuries that can cause ischemia-reperfusion injury includemyocardial infarct (MI) and stroke. The initiation of an inflammatoryresponse is likely caused by the increase in tissue oxygen levels thatoccur with reperfusion and the concomitant accumulation of metabolitesthat can generate oxygen free radicals which are immunostimulatory.Ischemia-reperfusion injury is associated with a variety of eventsincluding severity of myocardial infarction, cerebral ischemic events,intestinal ischemia, and many aspects of vascular surgery, cardiacsurgery, trauma, and transplantation. The injury is manifested byinflammatory events of the innate immune system, particularly activationof the complement system, in response to newly altered tissue asnon-self. As such ischemia-reperfusion injury is characterized by tissueedema caused by increased vascular permeability, and an acuteinflammatory cell infiltrate caused by influx of polymorphonuclearleukocytes.

Activation of the complement system plays a role in the inflammatoryevents of ischemia-reperfusion injury. The ischemia injury results inalterations of the cell membrane, affecting lipids, carbohydrates, orproteins of the external surface such that these exposed epitopes arealtered and can act as neo-antigens (modified self-antigens).Circulating IgM recognize and bind the neo-antigens to form immunecomplexes on the injured cell surface. The antigen-antibody complexesformed are classic activators of the classical pathway of complement,although all pathways are likely involved in some way to theexacerbating effects of the injury. The involvement of the classicalpathway of complement to ischemia-reperfusion injury is evidenced bymice genetically deficient in either C3 or C4 that display equalprotection from local injury in a hind limb and animal model of injury(Austen et al. (2003) Int J Immunopath Pharm 16:1). Conversely, in akidney model of ischemia injury, C3-, C5-, and C6-deficient mice wereprotected whereas C4-deficient mice were not, indicating the importanceof the alternative complement pathway (Guo et al. (2005) Ann Rev Immunol23:821). Mediators induced upon complement activation initiate aninflammatory response directed at the cell membrane at the site of localinjury.

A major effector mechanism of complement in ischemia-reperfusion injuryis the influx and activation of neutrophils to the inflamed tissue bycomplement components, such as, for example, C5a. Activation ofneutrophils results in increased production of reactive oxygen speciesand the release of lysosomal enzymes in local injured organs whichultimately results in apoptosis, necrosis, and a loss or organ function.The generation of the terminal MAC, C5b-9, also contributes to localtissue injury in ischemia-reperfusion injury.

f. Ocular Disorders

In the normal eye, the complement system is continuously activated atlow levels; membrane-bound and soluble intraocular complement regulatoryproteins tightly regulate this spontaneous complement activation. Lowlevel complement activation protects against pathogens without causingany damage to self-tissue and vision loss. The complement system andcomplement regulatory proteins control the intraocular inflammation inautoimmune uveitis and play an important role in the development ofcorneal inflammation, age-related macular degeneration and diabeticretinopathy. The complement system plays an important role in thepathogenesis of diabetic retinopathy (see, e.g., Ghosh et al. (2015)Endocr Rev 36:272-288) as well as diabetic neuropathy and diabeticcardiovascular disease. Spontaneous complement activation can causedamage to the corneal tissue after the infection. Complement inhibitionis a relevant therapeutic target in the treatment of various oculardiseases (see, e.g., Jha et al. (2007) Mol Immunol. 44:3901-3908).

Age-Related Macular Degeneration (AMD)

Age-related macular degeneration is a clinical term that describes avariety of diseases that are characterized by the progressive loss ofcentral vision. AMD is the leading cause of vision loss in agedindividuals in many industrialized countries (Jager et al. (2008) N EnglJ Med 358:2606-2617). Vision loss occurs due to the progressivedegeneration of the macula, the region at the back of the eye comprisinga high density of cone photoreceptors, which is specialized forhigh-acuity, central vision.

AMD can manifest as Dry (non-neovascular) AMD and/or Wet AMD. Dry AMD isthe more common (85-90% of cases) and milder form of AMD, and ischaracterized by small, round, white-yellow lesions (drusen) in andunder the macula. Advanced dry AMD, or geographic atrophy, leads tothinning of the retina due to loss of PRE photoreceptors, deteriorationof the macula and eventual blindness. Although rarer, vision lossassociated with wet AMD is generally more dramatic than in dry AMD. WetAMD includes the formation of pathogenic blood vessels, termed choroidalneovascularization (CNV), in which abnormal blood vessels developbeneath the retinal pigment epithelium (RPE) layer of the retina. CNVinvasion of the retina from the underlying choroid through fractures inBruch membrane, the extracellular matrix between the choroid and theretinal pigment epithelium (RPE), or their breakage can cause visionloss in AMD (e.g., due to subretinal hemorrhage and/or scarring).

Early clinical hallmarks of AMD include thickening of the Bruch membraneand the appearance of drusen (Gass, J. D. (1972) Trans. Am. Ophthalmol.Soc. 70: 409-36), which are extracellular lipoproteinaceous depositsconsisting of aggregated proteins, such as albumin, apolipoprotein E(APOE), components of the complement pathway (e.g., complement factor H(CFH), C1q, C3, C5, C5b, C6, C7, C8, C9, and vitronectin (Hageman etal., (2001) Prog. Retin. Eye. Res. 29:95-112; Hageman et al. (2005)Proc. Nat. Acad. Sci. 102: 7227-7232; Mullins et al. (2000) FASEB J14:835-846; Anderson et al., (2010) Prog. Retin. Eye Res. 29:95-112),immunoglobulins and/or amyloid-β (Crabb et al. (2002) Proc Natl Acad Sci99:14682-14687; Johnson et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99:11830-11835) and lipids and cellular components that are localizedbetween the RPE and the Bruch membrane. Inflammation in AMD is mediatedby the deregulation of the alternative complement pathway. Complementcomponents C3 and C5 are principal constituents of drusen in patientswith AMD (Mullins et al., (2000) FASEB J 14, 835-46; Johnson et al.,(2000) Exp Eye Res 70, 441-9; Anderson et al., (2002) Am J Ophthalmol134, 411-31; and Johnson et al., (2001) Exp Eye Res 73, 887-96). It ishypothesized that drusen biogenesis involves chronic inflammatoryprocesses that either can trigger complement activation and formation ofMAC, which can lyse RPE cells or disturb physiological homeostasis inRPE cells, leading to inflammation characteristic of AMD (Johnson et al.(2001) Exp Eye Res 73, 887-896). Complement proteins (e.g., C3d) alsowere detected in blood in AMD patients (Scholl et al., (2008) PLoS One3: e2593), indicating that AMD-induced inflammation can be systemic.There is genetic evidence for a role in complement in the pathogenesisof dry AMD (Klein et al. (2005) Science 308(5720):385-389; Yates et al.,(2007) NEJM 357:553-561) and compstatin (and compstatin derivativesAPL-1 and APL-2) and POT-4 (Potentia Pharmaceuticals), small peptideinhibitors of C3, may slow the progression of geographic atrophy(Ricklin et al. (2008) Adv. Exp. Med. Biol. 632: 273-292) in AMD,indicating that C3 (i.e., C3 inhibition) is a viable target for AMDtreatment. Recent clinical results have validated these conclusions andfindings. C3 is a viable clinical target for complement mediateddisorders and conditions or for those in which complement plays a role.

g. Organ Transplantation and Delayed Graft Function (DGF)

Complement plays a role in the pathogenesis of ischemia-reperfusioninjury. The mechanism of renal reperfusion injury depends on thegeneration of C5a and C5b-9, both of which have direct toxicity on therenal tubules contributing to acute tubular necrosis and apoptosis, andleading to post-ischemic acute renal failure and tissue fibrosis. Inturn, the generation of these terminal pathway components depends onintra-renal synthesis of C3 and availability of other complementcomponents that are essential for complement activation. The level ofexpression of C3 in the donor organ is strongly dependent on the coldischaemic time (Asgari et al. (2010) Curr Opin Organ Transplant.15:486-491).

Rejection in solid organ transplantation is influenced by the initialinflammatory response and subsequent adaptive alloimmune response, bothof which are affected by various complement components. Complementproteins play a significant part in organ damage followingtransplantation in the process of ischaemia reperfusion and inmodulating the activation of the adaptive immune response. Inhibitingcomplement or modulating the function of complement protein moleculescan reduce transplant organ damage and increase the organ lifespan (see,e.g., Asgari et al. (2010) Curr Opin Organ Transplant. 15:486-491).Targeting complement components for therapeutic intervention can reduceorgan damage at the time of organ recovery, transfer and aftertransplantation. Exemplary of such organs is the kidney. The modifiedMTSP-1 polypeptides provided herein can be administered to mitigateand/or treat organ damage following transplantation.

Delayed graft function (DGF), such as delayed graft function of thekidney, liver, lung, and/or heart, is a condition occurring in a subsetof organ transplant patients in which the transplanted organ fails tofunction normally immediately following transplant. Other possibletransplants include, but are not limited to, vascular tissue, eye,cornea, lens, skin, bone marrow, muscle, connective tissue,gastrointestinal tissue, nervous tissue, bone, stem cells, islets,cartilage, hepatocytes, and hematopoietic cells. Renal DGF ischaracterized by acute necrosis of the renal allograft and has beenclinically defined by the need for dialysis shortly followingtransplantation. Acute kidney injury during the transplant processfrequently manifests as DGF. The pathology underlying DGF is complexwith contributions from donor-derived factors such as donor age andduration of ischemia, and recipient factors such as reperfusion injury,immunological responses and treatment with immunosuppressantmedications.

Components of the complement cascade and complement activation play acritical role as mediators of transplant rejection andischemia-reperfusion injury leading to DGF. Animal studies haveestablished a key role for complement in ischemic reperfusion injury.For example, Eculizumab, a humanized monoclonal antibody directedagainst C5, blocks complement activation and was shown to preventdelayed graft function in a subset of high-risk kidney transplantpatients (see, e.g., Horizon Scanning Research and Intelligence Centrebrief, 2016 September; Johnson et al. (2015) Curr Opin Organ Transplant20(6):643-651; Yu et al. (2016) Am J Transplant 16(9):2589-2597).Granular C4d deposition was associated with DGF in human renal allograftrecipients (Kikie et al. (2014) Transpl Int 27(3):312-321). Increased C3production is associated with kidney transplant rejection (Pratt et al.(2002) Nat Med 8(6):582-587; Damman et al. (2011) Nephrol DialTransplant 26(7):2345-2354). Hence, the modified MTSP-1 polypeptidesprovided herein, can be used as a therapeutic for preventing orameliorating or eliminating transplant rejection and DGF.

2. Therapeutic Uses

a. Immune-Mediated Inflammatory Diseases

Modified MTSP-1 polypeptides described herein can be used to treatinflammatory diseases. Inflammatory diseases that can be treated withproteases include acute and chronic inflammatory diseases. Exemplaryinflammatory diseases include central nervous system diseases (CNS),autoimmune diseases, airway hyper-responsiveness conditions such as inasthma, rheumatoid arthritis, inflammatory bowel disease, and immunecomplex (IC)-mediated acute inflammatory tissue injury.

Experimental autoimmune encephalomyelitis (EAE) can serve as a model formultiple sclerosis (MS) (Piddlesden et al. (1994) J Immunol 152:5477).EAE can be induced in a number of genetically susceptible species byimmunization with myelin and myelin components such as myelin basicprotein, proteolipid protein and myelin oligodendrocyte glycoprotein(MOG). For example, MOG-induced EAE recapitulates essential features ofhuman MS including the chronic, relapsing clinical disease course, thepathohistological triad of inflammation, reactive gliosis, and theformation of large confluent demyelinated plaques. Modified MTSP-1polypeptides can be assessed in EAE animal models. Modified MTSP-1polypeptides are administered, such as by daily intraperitonealinjection, and the course and progression of symptoms is monitoredcompared to control animals. The levels of inflammatory complementcomponents that can exacerbate the disease also can be measured byassaying serum complement activity in a hemolytic assay and by assayingfor the deposition of complement components, such as for example C1, C3and C9.

Complement activation modulates inflammation in diseases such asrheumatoid arthritis (RA) (Wang et al., (1995) PNAS 92:8955). ModifiedMTSP-1 polypeptides can be used to treat RA. For example, MTSP-1polypeptides can be injected locally or systemically. Modified MTSP-1polypeptides can be dosed daily or weekly. PEGylated MTSP-1 polypeptidescan be used to reduce immunogenicity. In one example, type IIcollagen-induced arthritis (CIA) can be induced in mice as a model ofautoimmune inflammatory joint disease that is histologically similar toRA characterized by inflammatory synovitis, pannus formation, anderosion of cartilage and bone. To induce CIA, bovine type II collagen(B-CII) in the presence of complete Freund's adjuvant can be injectedintradermally at the base of the tail. After 21 days, mice can bere-immunized using the identical protocol. To examine the effects of aMTSP-1 polypeptide, 3 weeks following the initial challenge with B-CII,a MTSP-1 polypeptide or control can be administered intraperitoneallytwice weekly for 3 weeks. Mice can be sacrificed 7 weeks following theinitial immunization for histologic analysis. To assess the therapeuticeffect of a MTSP-1 polypeptide on established disease, a MTSP-1polypeptide can be administered daily for a total of 10 days followingthe onset of clinical arthritis in one or more limbs. The degree ofswelling in the initially affected joints can be monitored by measuringpaw thickness using calipers. In both models, serum can be drawn frommice for hemolytic assays and measurement of complement markers ofactivation such as for example C5a and C5b-9. In another example,primate models are available for RA treatments. Response of tender andswollen joints can be monitored in subjects treated with MTSP-1polypeptides and controls to assess MTSP-1 polypeptide treatment.

Modified MTSP-1 polypeptide can be used to treat immune complex(IC)-mediated acute inflammatory tissue injury. IC-mediated injury iscaused by a local inflammatory response against IC deposition in atissue. The ensuing inflammatory response is characterized by edema,neutrophilia, hemorrhage, and finally tissue necrosis. IC-mediatedtissue injury can be studied in an in vivo Arthus (RPA) reaction.Briefly, in the RPA reaction, an excess of antibody (such as for examplerabbit IgG anti-chicken egg albumin) is injected into the skin ofanimals, such as for example rats or guinea pigs, that have previouslybeen infused intravenously with the corresponding antigen (i.e., chickenegg albumin) (Szalai et al., (2000) J Immunol 164:463). Immediatelybefore the initiation on an RPA reaction, a MTSP-1 polypeptide, or abolus control, can be administered at the same time as the correspondingantigen by an intravenous injection via the right femoral vein.Alternatively, a MTSP-1 polypeptide can be administered during theinitial hour of the RPA reaction, beginning immediately after injectionof the antigen and just before dermal injection of the antibody. Theeffects of a MTSP-1 polypeptide on the generation ofcomplement-dependent IC-mediated tissue injury can be assessed atvarious times after initiation of RPA by collecting blood to determinethe serum hemolytic activity, and by harvesting the infected area of theskin for quantitation of lesion size.

Therapeutic MTSP-1 polypeptides, such as those described herein, can beused to treat sepsis and severe sepsis that can result in lethal shock.A model of complement-mediated lethal shock can be used to test theeffects of a MTSP-1 polypeptide as a therapeutic agent. In one suchexample, rats can be primed with a trace amount of lipopolysaccharide(LPS), followed by the administration of a monoclonal antibody against amembrane inhibitor of complement (anti-Crry) (Mizuno et al., (2002) IntArch Allergy Immunol 127:55-62). A MTSP-1 polypeptide or control can beadministered at any time during the course of initiation of lethal shocksuch as before LPS priming, after LPS priming, or after anti-Crryadministration and the rescue of rats from lethal shock can be assessed.

b. Neurodegenerative Disease

Complement activation exacerbates the progression of Alzheimer's disease(AD) and contributes to neurite loss in AD brains. Modified MTSP-1polypeptides described herein can be used to treat AD. Mouse models thatmimic some of the neuropathological and behavioral features of AD can beused to assess the therapeutic effects of MTSP-1 polypeptides. Examplesof transgenic mouse models include introducing the human amyloidprecursor protein (APP) or the presenilin 1 (PS1) protein withdisease-producing mutations into mice under the control of an aggressivepromoter. These mice develop characteristics of AD including increasesin beta-amyloid plaques and dystrophic neurites. Double transgenic micefor APP and PS1 mutant proteins develop larger numbers of fibrillarbeta-amyloid plaques and show activated glia and complement factorsassociated with the plaque. MTSP-1 polypeptides can be administered,such as by daily intraperitoneal or intravenous injections, and thecourse and progression of symptoms is monitored compared to controlanimals.

c. Cardiovascular Disease

Modified MTSP-1 polypeptides provided herein can be used to treatcardiovascular disease. MTSP-1 polypeptides can be used in the treatmentof cardiovascular diseases including ischemia reperfusion injuryresulting from stroke, myocardial infarction, cardiopulmonary bypass,coronary artery bypass graft, angioplasty, or hemodialysis. MTSP-1polypeptides also can be used in the treatment of the inflammatoryresponse associated with cardiopulmonary bypass that can contribute totissue injury. Generally, a MTSP-1 polypeptide can be administered priorto, concomitantly with, or subsequent to a treatment or event thatinduces a complement-mediated ischemia reperfusion injury. In oneexample, a MTSP-1 polypeptide can be administered to a subject prior tothe treatment of a subject by a complement-mediated, ischemic-injuryinducing event, such as for example coronary artery bypass graft orangioplasty.

Effects of a MTSP-1 polypeptide on treatment of ischemia reperfusioninjury can be assessed in animal models of the injury. In one suchmodel, myocardial ischemia is induced in rabbits that have had anincision made in their anterior pericardium by placing a 3-0 silk suturearound the left anterior descending (LAD) coronary artery 5-8 mm fromits origin and tightening the ligature so that the vessel becomescompletely occluded (Buerke et al., (2001) J Immunol 167:5375). A MTSP-1polypeptide, such as for example a modified MTSP-1 polypeptide, or acontrol vehicle such as saline, can be given intravenously in increasingdoses as a bolus 55 minutes after the coronary occlusion (i.e., 5minutes before reperfusion). Five minutes later (i.e., after a total of60 minutes of ischemia) the LAD ligature can be untied and the ischemicmyocardium can be reperfused for 3 hours. At the end of the reperfusionperiod, the ligature around the LAD is tightened. Effects of a MTSP-1polypeptide on ischemia injury can be analyzed by assessing effects onmyocardial necrosis, plasma creatine kinase levels, and markers ofneutrophil activation such as for example myeloperoxidase activity andsuperoxide radical release.

In another model of complement-mediated myocardial injury sustained uponperfusion of isolated mouse hearts with Krebs-Henseleit buffercontaining 6% human plasma, treatment with modified MTSP-1 polypeptidescan be used to limit tissue damage to the heart. In such an example, thebuffer used to perfuse the hearts can be supplemented with varying dosesof modified MTSP-1 polypeptides. The perfused hearts can be assayed fordeposition of human C3 and C5b-9, coronary artery perfusion pressure,end-diastolic pressure, and heart rate.

Modified MTSP-1 polypeptides provided herein can be used as therapeuticsprior to or following Cardiopulmonary Bypass (CPB) or coronary arterybypass graft to inhibit the inflammatory immune response that oftenfollows bypass and that can contribute to tissue injury. An in vitrorecirculation of whole blood in an extracorporeal bypass circuit can beused to stimulate platelet and leukocyte changes and complementactivation induced by CPB (Rinder et al. (1995) J Clin. Invest.96:1564). In such a model, addition of a MTSP-1 polypeptide or controlbuffer, in varying doses, can be added to a transfer pack alreadycontaining blood from a healthy donor and porcine heparin, just prior toaddition of the blood to the extracorporeal circuit. Blood samples canbe drawn at 5, 15, 30, 45, 60, 75, and 90 minutes after recirculationand assayed for complement studies such as for example hemolytic assaysand/or complement activation assays to measure for C5a, C3a, and/orsC5b-9. A pretreatment sample of blood drawn before its addition to theextracorporeal circuit can be used as a control. Flow cytometry of bloodsamples can be performed to determine levels of adhesion molecules onpopulations of circulating leukocytes (i.e. neutrophils) in the bloodsuch as for example CD11b and P-selectin levels.

d. Age-Related Macular Degeneration (AMD)

Modified MTSP-1 polypeptides described herein can be used to treatAge-Related Macular Degeneration (AMD). Age-Related Macular Degeneration(AMD) that can be treated with proteases include wet AMD, dry AMD andgeographic atrophy. Numerous animal models of AMD are available thatmimic many of the characteristics of the human disorder (Pennesi et al.(2012) Mol. Aspects Med. 33(4):487-509). Mutations in complement pathwaygenes were shown to increase or decrease susceptibility to AMD (Edwardset al. (2005) Science 308(5720):421-424; Hageman et al. (2005) Proc.Nat. Acad. Sci 102(20):7227-7232; Klein et al. (2005) Science308(5720):385-389). For example, in complement factor H (CFH), whichnormally interacts with C3b, the single nucleotide polymorphism Y402Hprevented binding of C3b with factor B, leading to inhibition of C3formation. Y402H is associated with an increased risk of AMD in peopleand the mutation was previously identified in 43-59% of AMD patients(Haines et al. (2005) Science 308(5720):419-421; Thakkinstian et al.(2006) Hum. Mol. Genet. 15(18):2784-2790; Zareparsi et al. (2005) Am. J.Hum. Genet. 77(1):149-153).

Genetically modified mice that lack the ability to make CFH developcharacteristics of AMD, including retinal abnormalities, decreasedvisual acuity and complement deposition (Coffey et al. (2007) Proc. Nat.Acad. Sci. 104:16651-16656). Mutations in complement proteins Factor B(Montes et al. (2009) Proc. Nat. Acad. Sci. 106(11):4366-4371), C2 (Goldet al. (2006) Nat. Genet. 38(4):458-462), and C3 (Maller et al. (2007)Nat. Genet. 39(10):1200-1201; Yates et al. (2007) New Engl. J. Med.357(6):553-561) are associated with increased or decreased risk ofdeveloping AMD based on their impact on expression and/or activity ofthe various complement proteins (Reynolds et al. (2009) Invest.Ophthalmol. Vis. Sci. 50(12):5818-5827).

Modified MTSP-1 proteases, such as modified MTSP-1 proteases providedherein, wherein an activity, such as substrate specificity orselectivity, of the MTSP-1 protease for cleaving complement protein C3is altered can be used as therapeutics. The modified MTSP-1 polypeptidesprovided herein are administered, for example, by monthly or bi-monthlyintravitreal injection, and the course and progression of symptoms ismonitored compared to control animals or subjects. The levels ofcomplement components that can exacerbate the disease also can bemeasured by assaying serum complement activity in a hemolytic assay andby assaying for the deposition of complement components, such as forexample C1, C3 and C9.

Complement activation plays a role in disease progress in Age-RelatedMacular Degeneration (AMD) (see e.g., Bradley et al., (2011) Eye25:683-693; Gemenetzi et al. (2016) Eye 30:1-14). Modified MTSP-1polypeptides can be used to treat AMD. For example, MTSP-1 polypeptidesor a pharmaceutical composition containing MTSP-1 polypeptides, such asthe modified MTSP-1 polypeptides described herein, can be injectedintravitreally or periocularly. Modified MTSP-1 polypeptides can bedosed daily or weekly or less frequently, such as for example, monthlyor less frequently, such as bi-monthly. For AMD, modified MTSP-1polypeptides that are further “modified” for extended duration in theeye (e.g., fusion proteins, PEGylation, etc.) monthly dosing can beused. Also, depending upon the particular modification, bi-monthly andtri-monthly dosing (every 3 months) also are contemplated. The modifiedMTSP-1 polypeptides can be modified, such as by PEGylation, to reducepotential immunogenicity and/or to increase serum half-life. For AMID,modified MTSP-1 polypeptides that are not further modified for extendedduration in the eye (e.g., fusion proteins, PEGylated proteins) monthlyor bi-monthly administration is contemplated. If modified, such as byPEGylation, dosing can be effected every 3 months or more.

e. Organ Transplant

Delayed Graft Function

Modified MTSP-1 polypeptides described herein can be used to treatDelayed Graft Function (DGF), including DGF, such as, for example, DGFas a result of Ischemia-Reperfusion Injury in kidney transplantrecipients. MTSP-1 polypeptides also can be used in the treatment of theinflammatory response associated with organ transplant that cancontribute to tissue injury. Generally, a MTSP-1 polypeptide can beadministered prior to, concomitantly with, or subsequent to a treatmentor event that induces a complement-mediated ischemia reperfusion injury.In one example, a MTSP-1 polypeptide can be administered to a subjectprior to the treatment of a subject by a complement-mediated,ischemic-injury inducing event, such as for example kidney transplant orkidney allograft.

Effects of a MTSP-1 polypeptide on treatment of delayed graft function,for example delayed graft function as a result of ischemia-reperfusioninjury, can be assessed in animal models of the injury, which mimiccharacteristics displayed in human kidney allografts or transplants.

The presence of early biomarkers of early graft dysfunction leading toDGF, including biomarkers for tubular epithelial cell injury canindicate the need for therapeutics. Biomarkers of DGF (i.e., serumcreatine) have been identified (Malyszko et al. (2015) Nature ScientificReports 5:11684; Wanga et al. (2015) PLoS One 10(9):e0136276). Earlydetection of biomarkers for DGF and therapeutic intervention, such as,for example, therapeutic treatment with a modified MTSP-1 polypeptide,can improve clinical outcomes.

Complement activation modulates disease progress in disorders such asdelayed graft function after organ transplant, for example kidneytransplant (Yu et al. (2016) Am J of Transplantation 16(9):2589-2597).Modified MTSP-1 polypeptides can be used to treat DGF. For example,MTSP-1 polypeptides can be administered for systemic delivery or can beinjected directly into the graft or the surrounding tissues. ModifiedMTSP-1 polypeptides can be administered prior to, during or aftertransplant. Modified MTSP-1 polypeptides can be dosed daily or weekly orless frequently, such as for example, monthly or less frequently, suchas bi-monthly. In some instances a single systemic dose of the modifiedMTSP-1 polypeptide is administered. Multiple infusions of the modifiedMTSP-1 polypeptide over several hours are also considered. ModifiedMTSP-1 polypeptides can be delivered chronically, if needed, forexample, the modified MTSP-1 polypeptides, such as the modified MTSP-1polypeptides described herein, can be delivered on a daily basis or onanother schedule to maintain an effective amount in the allograftrecipient. Modified MTSP-1 polypeptides can be used to prolong allograftsurvival in a recipient, in particular, chronic survival of theallograft. PEGylated MTSP-1 polypeptides can be used to reduceimmunogenicity.

3. Combination Therapies

MTSP-1 polypeptides provided herein can be used in combination withother existing drugs and therapeutic agents to treat diseases andconditions. Such treatments can be performed in conjunction with otheranti-inflammatory drugs and/or therapeutic agents. Examples ofanti-inflammatory drugs and agents useful for combination therapiesinclude non-steroidal anti-inflammatory drugs (NSAIDs) includingsalicylates, such as aspirin, traditional NSAIDs such as ibuprofen,naproxen, ketroprofen, nabumetone, piroxicam, diclofenac, orindomethacin, and Cox-2 selective inhibitors such as celecoxib (soldunder the trademark Celebrex®) or Rotecoxin (sold under the trademarkVioxx®). Other compounds useful in combination therapies includeantimetabolites such as methotrexate and leflunomide, corticosteroids orother steroids such as cortisone, dexamethasone, or prednisone,analgesics such as acetaminophen, aminosalicylates such as mesalamine,and cytotoxic agents such as azathioprine (sold under the trademarkImuran®), cyclophosphamide (sold under the trademark Cytoxan®), andcyclosporine A. Additional agents that can be used in combinationtherapies include biological response modifiers. Biological responsemodifiers can include pro-inflammatory cytokine inhibitors includinginhibitors of TNF-alpha such as etanercept (sold under the trademarkEnbrel®), infliximab (sold under the trademark Remicade®), or adalimumab(sold under the trademark Humira®), and inhibitors of IL-1 such asanakinra (sold under the trademark Kineret®). Biological responsemodifiers also can include anti-inflammatory cytokines such as IL-10, Bcell targeting agents such as anti-CD20 antibodies (sold under thetrademark Rituximab®), compounds targeting T antigens, adhesion moleculeblockers, chemokine receptor antagonists, kinase inhibitors such asinhibitors to mitogen-activated protein (MAP) Kinase c-Jun N-terminalKinase (JNK), or nuclear factor (NF)κB, and peroxisomeproliferator-activated receptor-gamma (PPAR-γ) ligands. Additionalagents that can be used in combination therapies includeimmunosuppressants. Immunosuppressants can include tacrolimus or FK-506;mycophenolic acid; calcineurin inhibitors (CNIs); CsA; sirolimus orother agents known to suppress the immune system.

MTSP-1 polypeptides provided herein also can be used in combination withagents that are administered to treat cardiovascular disease and/oradministered during procedures to treat cardiovascular disease such asfor example those described herein that contribute to inflammatoryconditions associated with complement-mediated ischemia-reperfusioninjury. For example, MTSP-1 polypeptides provided can be administered incombination with anti-coagulants. Examples of exemplary anti-coagulantsinclude, but are not limited to, heparin, warfarin, acenocoumarol,phenindione, EDTA, citrate, oxalate, and direct thrombin inhibitors suchas argatroban, lepirudin, bivalirudin, and ximelagatran.

MTSP-1 polypeptides provided herein also can be used in combination withagents that are administered to treat DGF. MTSP-1 polypeptides providedherein can, for example, be administered in combination with animmunosuppressive agent. Such combination is useful in prolongingallograft survival in a recipient, in particular, chronic survival ofthe allograft. In preferred embodiments, the combination is formulatedand prepared such that it is suitable for chronic administration to therecipient of the allograft, for example, stable formulations areemployed. In certain embodiments, the combination is formulated andprepared such that it is suitable for concurrent administration of themodified MTSP-1 polypeptides and the immunosuppressive drug to therecipient of the allograft. In certain embodiments, the combination isformulated and prepared such that it is suitable for sequential (ineither order) administration of the modified MTSP-1 polypeptides and theimmunosuppressive drug to the recipient of the allograft.

MTSP-1 polypeptides provided herein also can be used in combination withagents that are administered to treat macular degeneration. For example,modified MTSP-1 poly peptides can be administered with any one or moreof ranibizumab (sold under the trade name Lucentis™); bevacizumab (soldunder the trade name Avastin™); pegaptanib sodium (sold under the tradename Macugen™); aflibercept (sold under the trade name Eylea™); andverteporfin (sold under the trade name Visudyne™). MTSP-1 polypeptidesprovided herein also can be used in combination with an implantabletelescope, laser treatment or laser photocoagulation, surgery, and/orphotodynamic therapy, alone or in combination with the therapeuticverteporfin, to treat macular degeneration.

Additional agents, such as other complement inhibitors, can be used asanti-inflammatory drugs in combination therapy with modified MTSP-1polypeptides as described herein. Examples of such other complementinhibitors include cobra venom factor (CVF), polyanionic molecules suchas heparin, dextran sulphate, polyvinyl sulphate, polylysine, orsuramin, natural molecules such as K-76COOH, Rosmarinic acid, or extractof the Chinese medicinal herb Ephedra, synthetic molecules such asnafamostat mesilate (FUT-175), a synthetic inhibitor of C1s(C1s-INH-248), or an inhibitor against C1s and fD (BCX-1470), peptideinhibitors such as compstatin, antibody inhibitors of complement such asanti-C5 (N19-8), a humanized anti-C5 (h5G1.1), anti-C6, or anti-C8antibodies, and soluble forms of membrane complement regulators such assoluble CR1 (sCR1), soluble DAF (sDAF), soluble MCP (sMCF), or solubleCD59 (sCD59) (Morgan et al., (2003) Mol Immunol. 40:159).

Pharmaceutical compositions containing MTSP-1 polypeptides describedherein can be used to treat any one or more inflammatory diseases orconditions mediated by complement activation. Also provided arecombinations of MTSP-1 polypeptides and another treatment or compoundfor treatment of an inflammatory disease or condition. The MTSP-1polypeptides and the anti-inflammatory agent can be packaged as separatecompositions for administration together or sequentially orintermittently. Alternatively, they can be provided as a singlecomposition for administration or as two compositions for administrationas a single composition. The combinations can be packaged as kits,optionally with additional reagents, instructions for use, vials andother containers, syringes and other items for use of the modifiedMTSP-1 polypeptides.

I. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Cloning and Expression of Modified MTSP-1 Polypeptides andScreening for Modified MTSP-1 Polypeptides that Cleave C3 in a TargetSite

A. Cloning and Mutagenesis of MTSP-1

A nucleic acid encoding amino acids 615-855 with the C122S replacementof the human MTSP-1 polypeptide set forth in SEQ ID NO: 1 was prepared.The construct included the pro-region, activation sequence, and proteasedomain, and contained residues 598 to the C-terminus of the sequencepublished by Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A.96:11054 and SEQ ID NO:1 (i.e., corresponding to residues 598 to 855 ofthe sequence of amino acids set forth in SEQ ID NO:1).

Modified MTSP-1 polypeptides were generated by Quikchange® site directedmutagenesis (Stratagene) according to the manufacturer's instructionswith specifically designed oligonucleotides that served as primers toincorporate designed mutations into the newly synthesized DNA. Briefly,a PCR sample reaction was set up containing the wild type MTSP-1 as atemplate and oligonucleotide primers designed to contain the desiredmutation(s). Following PCR, each reaction product was digested with DpnIto remove dam methylated parental strands of DNA. The DNA was thentransformed into E. coli XL-1 Blue Supercompetent cells (Stratagene) andplated on selective agar containing 50 μg/ml carbenicillin. Plasmid DNAwas isolated from selected clones, and sequenced to verify incorporationof mutation(s) at the desired location(s) within the MTSP-1 gene and theabsence of any additional, undesired mutations.

B. Preparation of MTSP-1 Polypeptides

1. Transformation

The protease domain of the wild-type and modified MTSP-1 polypeptides(both containing the C122S replacement) as detailed in Section A, above,were cloned into the pQE-80 expression vector (Qiagen) and the resultingconstructs transformed into BL21 Gold (DE3) E. coli cells (AgilentTechnologies, Catalog number: 230132). Approximately 50 μL of chemicallycompetent BL21 Gold (DE3) cells were transformed with the appropriateplasmid DNA (typically approximately 5 ng of purified plasmid DNA or5-10 μL of a ligation reaction mixture). Cells and DNA were incubated onice for 30 minutes, heat shocked at 42° C. for 45 sec, and thenincubated on ice for 2 minutes. 450 μL of room temperature TerrificBroth (TB media) (VWR International, Catalog number: 100219-866) wereadded, and the cells were grown in the TB media for 1 hour with shakingat 240 rpm at 37° C. 20 μL of solution containing the transformed cellswere spread on a 2×YT medium+100 μg/mL carbenicillin plate from Teknova(Catalog number: Y4420) and incubated overnight at 37° C. Isolatedcolonies were then selected and used for plasmid preparations. Theresulting plasmids were subjected to DNA sequencing to confirm thepresence of the coding sequence for the desired MTSP-1 polypeptide.

2. Expression of MTSP-1 Polypeptides

Five hundred μl of an overnight culture of cells that had beentransformed with a pQE-80 expression plasmid containing the codingsequence for the desired MTSP-1 polypeptide were added to 100 mL ofTerrific Broth/Carbenicillin₁₀₀ containing 2.1 mL of Solution 1, 5.4 mLof Solution 2, and 107 μl of Solution 3 from Overnight Express™Autoinduction System 1 in a 500 mL Erlenmeyer flask. The flask wasplaced into an Infors Multitron Shaker set at 210 rpm. The culture wasgrown (with shaking) overnight at 37° C. The following morning, 20 μl of1M Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the cultureto induce expression of the MTSP-1 polypeptide, and the culture wasgrown at 37° C. with shaking for an additional 2 hours. This “inducedculture” was collected in a 250 mL conical centrifuge bottle and spun at3600 rpm for 10 min at 4° C. in an Allegro™ 6R centrifuge with aGH-3.8/GH-3.8A rotor. The resulting cell pellet was either stored at−20° C. or further processed immediately as described below.

3. Isolation of MTSP-1 Polypeptide Inclusion Bodies

The bacterial cell pellet from the 100 mL culture was resuspended in 60ml BugBuster® extraction reagent (Merck Millipore, NC9591474) containing60 μL rLysozyme™ (Sigma, Catalog number: L6876) by vortexing followed byshaking at 240 rpm for 1 hr at 37° C. to facilitate lysis of thebacteria. After bacterial lysis, insoluble material was pelleted bycentrifugation at 3,600 rpm for 10 minutes at 4° C., and the supernatantwas decanted. The resulting pellet was resuspended by homogenizing in100 mL of 50 mM Tris pH 8.0 using a Power Gen 500 homogenizer (FisherScientific, 14-261-04P) with a 20×195 mm generator probe using 2×5second pulses repeated until the pellet was well dispersed. Theresuspended material was centrifuged at 3,600 rpm for 10 minutes at 4°C., supernatant decanted, and the pellet was allowed to air dry for 10to 15 minutes. The pellet of inclusion bodies (TB) was weighed andstored at −20° C. until use.

4. Resuspension and Unfolding of Inclusion Bodies Containing MTSP-1Polypeptides

The insoluble MTSP-1 polypeptides were isolated from inclusion bodiesand denatured in the presence of reducing agent. 4-5 grams of the IBpellet were resuspended in 25 mL of 50 mM Tris, pH 8.0 by homogenizationto form the IB solution. 75 mL of unfolding buffer (6M GuHCl, 50 mM TrispH 8.0, (Teknova, Catalog number: G0380)) was added to the D3 solution.The resulting IB solution was agitated at 240 rpm at 37° C. for at least1 hour, or until the inclusion bodies were fully dissolved.

5. Refolding of MTSP-1 Polypeptides

Five ml of the resuspended, denatured MTSP polypeptide solutiondescribed above was dripped slowly into ≥100 ml of Refolding Buffer (1.5M Arginine, 50 mM Tris pH 7.5, 150 mM NaCl) at a dilution of 1:20 orgreater (or approximately ≤100 protein/ml refolding buffer), withstirring. The protein solution in Refolding Buffer was incubated on ashaker at 150 rpm for 24 hours at room temperature to allow folding totake place.

The resulting protein solution was transferred to 12,000-14,000 Daltonmolecular weight cutoff (MWCO) Spectra/Por® regenerated cellulosedialysis tubing (VWR) and dialyzed in 180 L of 25 mM Tris, pH 8.0 for atleast 4 hours. The following day, the samples were transferred to a newtank of 180 L of 25 mM Tris, pH 8.0 and allowed to dialyze overnight.The following day, the samples were transferred to a third tank of 180 Lof 25 mM Tris, pH 8.0 and dialyzed for at least 18 hours. Samples weredialyzed at least overnight, and for up to multiple days. Samplesdialyzed for one day were incubated at room temperature and samplesdialyzed for more than one day were incubated at 4° C. The ratio oftotal dialysis buffer volume to total sample volume was at least 100.Following dialysis, the protease samples were removed from the dialysistubing and filtered using a 500 mL, 0.22 μm flask (Millipore), and theconductivity of the solution was measured. The conductivity of thesolution was adjusted to prevent non-specific binding to the Benzamidinecolumn during the activation step described below. The NaClconcentration was adjusted to approximately 0.5 M NaCl (e.g., 390 mL of5 M NaCl was added to 3.9 L of dialyzed protein). The conductivity ofthe solution should be approximately 1.3 ms (/cm).

6. Activation of MTSP-1 Polypeptides

After filtration of the solution containing refolded MTSP-1 polypeptide,the NaCl concentration was adjusted to approximately 0.5 M by additionof 5M NaCl. Twenty mL of immobilized trypsin agarose beads were packedinto an Econo-Pak disposable column (Bio-Rad; Catalog number 7321010)and the column was equilibrated with 200 mL (i.e., 10 column volumes) ofChromatography Solution Buffer A (25 mM Tris pH 8.0, 0.5M NaCl). Therefolded MTSP-1 polypeptide solution (see above) was loaded on thecolumn, and the flow throw containing the activated MTSP-1 polypeptidewas collected. The flow through fractions that contained protein werecombined and dialyzed overnight in 25 mM Tris buffer (pH=8.0) and bufferexchanged twice in 150 liters. The conductivity of the resulting,activated MTSP-1 polypeptide solution was confirmed to be less than 2ms/cm, or adjusted appropriately with 25 mM Tris, pH=8.0.

7. Purification of MTSP-1 Polypeptides

The MTSP-1 polypeptides can be and have been purified according to thefollowing steps:

-   -   1. Measure the conductivity of the activated sample using a        SevenEasy Conductivity Meter. If the measurement is <3 ms/cm,        then proceed to step 2 below. If the measurement is >3 ms/cm,        dilute sample with Chromatography Solution Buffer A (25 mM Tris        pH 8.0, 0.5M NaCl) until conductivity reaches <3 ms/cm.    -   2. Using an AKTAPURIFIER, load sample onto a 5 mL HiTrap Q HP        cation exchange column pre-equilibrated with 5 column        volumes (CV) of Chromatography Solution Buffer A at a flow rate        of 8 mL/min.    -   3. Wash the column with 5 column volumes of Chromatography        Solution Buffer A.    -   4. Elute with 15 column volumes of 0-50% NaCl gradient using        Chromatography Solutions Buffer A/Buffer B at 5 mL/min. Collect        2 ml fractions.    -   5. The column is washed with 3 CV of Chromatography Solution        Buffer B, and then re-equilibrated with 5 CV of Chromatography        Solution Buffer A prior to loading of the next sample.    -   6. Active MTSP-1 is located by activity assay, where 5 μl of        fraction is mixed with 50 μl of Assay Buffer containing 200 μM        of an appropriate quenched fluorescence substrate (add QHAR QF        substrate).    -   7. Read activity on a Molecular Devices M5 plate reader at        30° C. Set Excitation at 490 nm and Emission at 520 nm.    -   8. Concentrate the four most active fractions using a 15 mL        Amicon Ultra 10K Centrifugal Filter.    -   9. Measure concentration by A₂₈₀ using the Nanodrop        Spectrophotometer. Continue concentrating until achieving an        approximate final concentration of 200 μM.    -   10. To assess quality of purified product, load 2 μg/lane of        sample in 2× Sample Buffer containing 2× NuPAGE Sample Reducing        Agent on a 4-12% NuPAGE Novex Bis-Tris gel. Run the gel in 1×MES        Running Buffer at 200V for 30 min. Visualize the gel by staining        with Coomassie Blue followed by destaining.    -   11. Snap freeze the sample in liquid nitrogen and store at −80°        C.

8. Endotoxin Removal from Purified MTSP-1 Polypeptides for Use in InVivo Models

Removal of high molecular weight Endotoxin was achieved by passing thesample through a 15 ml Amicon Ultra-15 Centrifugal Filter Unit 100Kmembrane (Fisher Scientific). The sample was filtered by spinning at4000 RPM for 20 minutes. If sample had not passed through the filterafter 20 minutes, the centrifugation step was repeated. Then, the samplewas transferred to a clean falcon tube. To ensure complete removal ofsmall molecular weight Endotoxin, the sample was passed over a 2 mlCellufine ET clean S gravity column (Fisher Scientific). At least 24hours prior to addition of the sample, the 2 ml Cellufine ET clean Scolumn was prepared. 4 mL of Cellufine ET clean S slurry and 10 mLEndo-Free 20% Ethanol solution (Fisher Scientific) was added to theEcono-Pac Chromatography Gravity Column. The 20% EtOH solution waspassed through the column, followed by 25 mL of 80% EtOH, 0.2N NaOH. Thebottom of the gravity column was capped and the column was filled with80% EtOH, 0.2N NaOH, covered and incubated at room temp for at least 16hours. After at least 16 hours, the 80% EtOH, 0.2N NaOH was allowed topass through the column. The column was rinsed with 25 mL endotoxin-freewater and the water was allowed to pass through the column. Next, thecolumn was equilibrated with 2×20 mL sterile endo-free phosphatebuffered saline (PBS). The sample was then added to the column and theeluted sample was collected. The column was washed with 10 mL endo-freePBS to ensure that the sample had completely passed through the column.

Protein concentration, A₂₈₀, was measured using a NanoDrop®Spectrophotometer (NanoDrop) and use of a theoretical extinctioncoefficient of 2.012 mg/A₂₈₀. If necessary, active protein wasconcentrated using 15 mL Amicon Ultra-15 10K membrane Centrifugal FilterUnits (Fischer Scientific) to approximately 2.5 mg/ml incitrate-buffered saline (20 mM Sodium Citrate pH 5.0, 50 mM NaCl). Toassess the quality of the purified product, 2 μg of sample in 1× NuPAGELDS Sample buffer (Invitrogen, Corp.) containing 2× NuPAGE samplereducing agent (Invitrogen) was loaded on a 12-well 4-12% NuPAGE Novex4-12% Bis-Tris Gel (Invitrogen), and run in 1× NuPAGE MES SDS RunningBuffer (Invitrogen) at 200 V for 30 minutes. The cell was stained withCoomassie Blue followed by destaining to visualize protein bands.Protein was snap-frozen in liquid nitrogen and stored at −80° C.

C. Selection and Identification of Modified MTSP-1 Polypeptides ThatCleave C3 to Inactivate it

Modified MTSP-1 polypeptides were identified by screening a library ofmodified MTSP-1 polypeptides against a modified serpin ATIII asdescribed in detail in U.S. Pat. No. 8,211,428 (see, also U.S.Publication No. US-2014-0242062-A1, now U.S. Pat. No. 9,795,655). Aninhibitory serpin, or fragment thereof, capable of forming a covalentacyl enzyme intermediate between the serpin and protease is used forscreening. Generally, the serpin that is used is one that targets andregulates the wild type protease in vivo. Any serpin, however, thatreacts with and irreversibly inhibits the target protease can be used asa “bait” for these selection experiments.

In the assay, a serpin modified by replacement of its reactive site loop(RSL) to include a target sequence (i.e., the target site in C3 forcleavage to inactive C3) captures modified proteases that cleave thetarget site to form stable complexes. The captured modified protease isthen isolated/identified. For the MTSP-1 polypeptides, the “bait” serpinwas ATIII that was modified by replacing the residues indicated belowwith QHARASHLG (residues 737-745 of C3, SEQ ID NO: 9), which is thetargeted site for inactivation of human C3.

ATIII “bait” (SEQ ID NO: 692) with inserted sequence QHARASHLGcorresponding to the targeted site of C3 (residues 737-745 of SEQ IDNO:9):

        10         20         30         40HGSPVDICTA KPRDIPMNPM CIYRSPEKKA TEDEGSEQKI         50         60         70         80PEATNRRVWE LSKANSRFAT TFYQHLADSK NDNDNIFLSP        90        100        110        120LSISTAFAMT KLGACNDTLQ QLMEVFKFDT ISEKTSDQIH       130        140        150        160FFFAKLNCRL YRKANKSSKL VSANRLFGDK SLTFNETYQD       170        180        190        200ISELVYGAKL QPLDFKENAE QSRAAINKWV SNKTEGRITD       210        220        230        240VIPSEAINEL TVLVLVNTIY FKGLWKSKFS PENTRKELFY       250        260        270        280KADGESCSAS MMYQEGKFRY RRVAEGTQVL ELPFKGDDIT       290        300        310        320MVLILPKPEK SLAKVEKELT PEVLQEWLDE LEEMMLVVHM       330        340        350        360PRFRIEDGFS LKEQLQDMGL VDLFSPEKSK LPGIVAEGRD       370        380        390        400DLYVSDAFHK AFLEVNEEGS EAAASTAVV Q HARASHLG RV       410        420        430        440TFKANRPFLV FIREVPLNTI IFMGRVANPC VKGGGSDYKD DDDKThe mutations in the ATIII with reference to the position in SEQ ID NO:692 are summarized as follows:

RCL QHARASHLG 390 398 C-Terminus GGGSDYKDDDDK 433 444 Mutation I390Q 390390 Mutation A391H 391 391 Mutation G392A 392 392 Mutation S394A 394 394Mutation L395S 395 395 Mutation N396H 396 396 Mutation P397L 397 397Mutation N398G 398 398 Flag Tag DYKDDDDK 437 444

In order to perform the selection for protease variants which aretrapped by the QHAR-ASHLG modified ATIII bait, a library of MTSPvariants was displayed on the surface of M13 bacteriophage, fused to theC-terminus of phage coat protein P3. Several MTSP libraries weredesigned by substituting the natural codons with NNK codons at positionsin MTSP hypothesized to be important for substrate recognition andcleavage based on molecular modeling or were “second sphere” positionsthat contact mutated residues that were previously selected and provedto be advantageous. These positions corresponded to both prime side ornon-prime side sites on MTSP, including positions 40, 41, 60b, 60g,96-99 (plus insertions of 1 and 2 amino acids within the 96-99 region),151, 175, 192, 217, and 224. To ensure sufficient representation foreach variant in the phage library, the size of each constructed library(measured by CFUs generated of transformed library DNA) wassignificantly greater than the calculated diversity for all librariesthat contained 5 or 6 randomized positions and comparable to thecalculated diversity for libraries that contained 7 randomizedpositions. After construction of the MTSP phage libraries, 96 coloniesfrom each library were sequenced to confirm mutation frequency anddistribution and to assess the overall library quality. High qualitylibraries (i.e., those containing the expected frequency of mutations,no contamination, etc.) were then subjected to selection by incubatingwith the multiple concentrations of the biotinylated bait serpin forvarious lengths of time. The trapped biotinylated-MTSP-phage complex wascaptured on avidin coated plates, washed with 6M Guanidine hydrochlorideto remove high affinity, non-covalent protease-serpin complexes, andthen eluted with DTT. On some occasions a counterselection, using alpha2-macroglobulin or naturally occurring serpins, such as ATIII wasperformed. Frequently, the selection and counterselection were performedsimultaneously by incubating the library with both target serpin andcounterselection serpin(s) in the same reaction. Typically, thecounterselection serpin(s) would be present in molar excess over theselection serpin. Several rounds of selection were performed, thenindividually outgrown colonies were screened by enzyme assay forperformance against a peptide corresponding to the target substrate.Colonies producing variants with high activity for cleavage of thetarget sequence were further characterized by DNA sequencing of theirphagemid DNA to identify the identity of the mutations in the MTSP-1coding sequence.

D. Modified MTSP-1 Polypeptides

Table 14 below sets forth modifications and the sequences of exemplaryprotease domains of modified MTSP-1 polypeptides that were generated andselected to inactivate C3, with the mutations indicated using numberingrelative to the mature MTSP-1 polypeptide set forth in SEQ ID NO:1(mature MTSP-1 numbering), and also chymotrypsin numbering (additionalmodifications are set forth in Table 15). While the SEQ ID NOs.reference protease domains, it is understood that the mutations can beincluded in mature modified MTSP-1 polypeptides, catalytically activeportions thereof that contain the referenced modifications, and activeforms and activated two chain forms.

The C122S replacement, or other conserved replacement for S, is includedto eliminate dimerization of the protease and reduce the potential forformation of “inappropriate” disulfide bonds during the folding process;while advantageous, it is an optional mutation.

TABLE 14 Modified MTSP-1 Polypeptides SEQ ID Mature MTSP-1 numberingChymotrypsin numbering NO.* I640R/F706T/InsE/T707G/I41R/F97T/Ins97aE/T98G/ 21 F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802E Q192E Q637H/I640A/D661V/F664R/Q38H/I41A/D60bV/F60eR/ 22 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802D Q192D Q637H/I640A/D661T/F664K/Q38H/I41A/D60bT/F60eK/ 23 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802D Q192D Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 24 Y666W/F706D/InsV/T707P/Y60gW/F97D/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802E Q192E Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 25 Y666W/F706D/InsV/T707P/Y60gW/F97D/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640A/D661T/F664K/Q38H/I41A/D60bT/F60eK/ 26 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 27 Y666W/F706D/InsV/T707P/Y60gW/F97D/ins97aV/T98P/ F708L/C731S/G759N/Q783L/F99L/C122S/G151N/Q175L/ Q802D Q192D Q637H/I640A/D661V/F664R/Q38H/I41A/D60bV/F60eR/ 28 Y666W/F706T/InsE/T707G/Y60gW/F97T/ins97aE/T98G/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640A/D661V/F664R/Q38H/I41A/D60bV/F60eR/ 29 Y666W/D705I/F706Y/InsN/Y60gW/D96I/F97Y/ins97aN/ T707G/F708L/C731S/G759N/ T98G/F99L/C122S/G151N/Q783L/Q802D Q175L/Q192D Q637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/30 Y666W/D705K/F706D/InsA/ Y60gW/D96K/F97D/ins97aA/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640A/D661V/F664R/ Q38H/I41A/D60bV/F60eR/ 31Y666W/D705P/F706W/InsN/ Y60gW/D96P/F97W/ins97aN/T707G/F708L/C731S/G759N/ T98G/F99L/C122S/G151N/ Q783L/Q802E Q175L/Q192EQ637H/I640A/D661V/F664R/ Q38H/I41A/D60bV/F60eR/ 32Y666W/D705I/F706N/T707G/ Y60gW/D96I/F97N/T98G/F99L/F708L/C731S/G759N/Q783L/ C122S/G151N/Q175L/Q192D Q802DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 33Y666W/D705Y/F706E/InsV/ Y60gW/D96Y/F97E/ins97aV/T707G/F708L/C731S/G759H/ T98G/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 34Y666W/D705L/F706D/InsG/ Y60gW/D96L/F97D/ins97aG/T707N/F708L/C731S/G759H/ T98N/F99L/C122S/G151H/ Q783L/Q802E Q175L/Q192EQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 35Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 36Y666W/D705V/F706G/InsV/ Y60gW/D96V/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 37Y666W/D705K/F706D/InsA/ Y60gW/D96K/F97D/ins97aA/T707P/F708L/C731S/G759N/ T98P/F99L/C122S/G151N/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 38Y666W/F706G/InsV/T707P/ Y60gW/F97G/ins97aV/T98P/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 39Y666W/D705K/InsV/T707P/ Y60gW/D96K/ins97aV/T98P/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 40Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q783L Q175LI640E/F708L/C731S/G759N/ I41E/F99L/C122S/G151N/ 41 Q802T Q192TI640D/C731S/G759N/Q802T I41D/C122S/G151N/Q192T 42I640S/F708L/C731S/G759N/ I41S/F99L/C122S/G151N/ 43 Q802V Q192VI640E/F708L/C731S/G759N/ I41E/F99L/C122S/G151N/ 44 Q802T Q192TI640D/Y658F/D705E/F708L/ I41D/Y59F/D96E/F99L/C122S/ 45 C731S/G759N/Q802TG151N/Q192T I640D/Y658F/C731S/G759N/ I41D/Y59F/C122S/G151N/ 46 Q802TQ192T I640S/D661T/F664S/Y666W/ I41S/D60bT/F60eS/Y60gW/ 47D705K/F706G/InsV/T707P/ D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/D661T/F664S/Y666W/Q38H/D60bT/F60eS/Y60gW/ 48 D705K/F706G/InsV/T707P/D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/Q802D Q192D Q637H/I640S/F664S/Y666W/ Q38H/I41S/F60eS/Y60gW/ 49D705K/F706G/InsV/T707P/ D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640S/D661T/Y666W/Q38H/I41S/D60bT/Y60gW/ 50 D705K/F706G/InsV/T707P/D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/Q802D Q192D Q637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 51D705K/F706G/InsV/T707P/ D96K/F97G/ins97aV/T98P/ F708L/C731S/G759H/Q783L/F99L/C122S/G151H/Q175L/ Q802D Q192D Q637H/I640S/D661T/F664S/Q38H/I41S/D60bT/F60eS/ 52 Y666W/D705K/F706G/T707P/ Y60gW/D96K/F97G/T98P/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 53Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/F708L/C731S/G759H/Q783L/ F99L/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 54Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/C731S/G759H/Q783L/ T98P/C122S/G151H/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 55Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/Q783L/ T98P/F99L/C122S/Q175L/ Q802D Q192DQ637H/I640S/D661T/F664S/ Q38H/I41S/D60bT/F60eS/ 56Y666W/D705K/F706G/InsV/ Y60gW/D96K/F97G/ins97aV/T707P/F708L/C731S/G759H/ T98P/F99L/C122S/G151H/ Q802D Q192DQ637H/I640S/D705K/F706G/ Q38H/I41S/D96K/F97G/ 57 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q802D Q192D I640S/D705K/F706G/InsV/I41S/D96K/F97G/ins97aV/ 58 T707P/F708L/C731S/Q783L/T98P/F99L/C122S/Q175L/ Q802D Q192D Q637H/I640S/D705K/F706G/Q38H/I41S/D96K/F97G/ 59 InsV/T707P/F708L/C731S/ ins97aV/T98P/F99L/C122S/Q783L/Q802D Q175L/Q192D I640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/63 T707P/F708L/C731S/Q802D T98P/F99L/C122S/Q192DQ637H/I640S/D705K/F706G/ Q38H/I41S/D96K/F97G/ 64 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D661Y/D705K/ Q38H/I41S/D60bY/D96K/ 65F706G/InsV/T707P/F708L/ F97G/ins97aV/T98P/F99L/ C731S/Q802D/D828VC122S/Q192D/D217V I640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/ 66T707P/F708L/C731S/Q802G/ T98P/F99L/C122S/Q192G/ D828V D217VI640S/D661Y/D705K/F706G/ I41S/D60bY/D96K/F97G/ 67InsV/T707P/F708L/C731S/ ins97aV/T98P/F99L/C122S/ Q802D/D828V Q192D/D217VI640S/D705M/F706G/InsV/ I41S/D96M/F97G/ins97aV/ 68T707P/F708L/C731S/Q802G/ T98P/F99L/C122S/Q192G/ D828V D217VI640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/ 69T707P/F708L/C731S/Q802V/ T98P/F99L/C122S/Q192V/ D828I D217II640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/ 70T707P/F708L/C731S/Q802H T98P/F99L/C122S/Q192H I640S/D705K/F706G/InsV/I41S/D96K/F97G/ins97aV/ 71 T707P/F708L/C731S/Q802N/T98P/F99L/C122S/Q192N/ D828V D217V I640S/D661Y/D705K/F706G/I41S/D60bY/D96K/F97G/ 72 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q783L/Q802D Q175L/Q192DQ637H/I640S/D705K/F706G/ Q38H/I41S/D96K/F97G/ 73 InsV/T707P/F708L/C731S/ins97aV/T98P/F99L/C122S/ Q802G/D828V Q192G/D217V I640S/D705K/F706G/InsV/I41S/D96K/F97G/ins97aV/ 74 T707P/F708L/C731S/Q783L/T98P/F99L/C122S/Q175L/ Q802V Q192V I640S/P648S/D705K/F706G/I41S/P49S/D96K/F97G/ 75 InsV/T707P/F708L/C731S/ ins97aV/T98P/F99L/C122S/Q802G/D828V Q192G/D217V I640S/D705K/F706G/InsV/ I41S/D96K/F97G/ins97aV/76 T707P/F708L/C731S/Q783L/ T98P/F99L/C122S/Q175L/ Q802N/D828VQ192N/D217V I640T/F706W/F708L/C731S/ I41T/F97W/F99L/C122S/ 77G759N/Q783M/Q802G/ G151N/Q175M/Q192G/ D828L D217LI640G/F706L/F708L/C731S/ I41G/F97L/F99L/C122S/ 78 Q783A/Q802T/D828VQ175A/Q192T/D217V I640G/F706V/F708L/C731S/ I41G/F97V/F99L/C122S/ 79G759Q/Q783M/Q802A/D828L G151Q/Q175M/Q192A/D217L I640G/F706I/F708L/C731S/I41G/F97I/F99L/C122S/ 80 G759L/Q783M/Q802S/D828V G151L/Q175M/Q192S/D217VI640G/F706S/F708L/C731S/ I41G/F97S/F99L/C122S/ 81G759N/Q783L/Q802G/D828I G151N/Q175L/Q192G/D217I *SEQ ID of the proteasedomain containing the replacements

Example 2 In Vitro Cleavage of Complement Protein C3

The activity of the modified MTSP-1 polypeptides was determined bycleavage of the substrate complement protein, human C3, by measuring theamount of intact human C3 remaining after incubation with variousconcentrations of the protease for 1 hour at 37° C. In this assay,signal is generated in the presence of intact human C3, and is lost asthe C3 is cleaved.

2 μM plasma purified human C3 (hC3; Complement Technologies; Tyler, TX)was incubated with the modified MTSP-1 polypeptides (0-250 nM) for 1hour at 37° C. in buffer containing 50 mM Tris, pH 8.0, 50 mM NaCl, and0.01% Tween-20. The activity of the modified MTSP-1 polypeptides wasquenched by the addition of EGR-CMK (Haematologic Technologies,EGRCK-01) to a final concentration of 10 μM and the hC3/modified MTSP-1polypeptide mixture was allowed to stand for 30 minutes at ambienttemperature.

Residual levels of undigested human C3 were quantified using anAmplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen®;Perkin Elmer). α-mouse IgG-coated acceptor beads at 100 μg/mL (PerkinElmer #6760606) were incubated with 5 nM mouse α-hC3a mAb (Abeam#ab11872-50) in 50 mM Tris, pH 8.0, 50 mM NaCl, 0.01% Tween-20 and 0.2%BSA to form the acceptor bead mixture. The acceptor bead mixture wasshielded from light and placed on a rotating shaker for 30-60 minutes.The hC3/modified MTSP-1 polypeptide reaction mixtures (prepared above)were diluted 1600-fold into 50 mM Tris, pH 8.0, 50 mM NaCl, 0.01%Tween-20, 0.2% BSA and 4 μL aliquots were placed in duplicate wells of a384-well Optiplate (Perkin Elmer #6007299). 8 μL of a α-hC3 mAb/acceptorbeads mixture was incubated with 8 μL of 25 nM biotinylated goat α-hC3pAb (prepared using EZ-Link Sulfo-NHS-LC-Biotin kit from ThermoScientific #21327 from the unbiotinylated version from ComplementTechnologies #A213). The plate was then shielded from light andincubated for 30 minutes at ambient temperature. After this incubation,4 μL of 100 μg/mL streptavidin-coated donor beads (Perkin Elmer#6760606) were added to each well and incubated for 60 minutes, shieldedfrom light. The alphascreen signal (Excitation=680 nm, Emission=570 nm)was then measured using an Envision 2104 Multilabel plate reader (PerkinElmer). This signal (corresponding to the concentration of remaining hC3([hC3])) was plotted as a function of modified MTSP-1 polypeptideconcentration ([Alterase]) and the data were fitted to the fourparameter equation below to determine the concentration of modifiedMTSP-1 polypeptide (the ‘alterase’ concentration) required to cleave 50%of the available hC3 (EC₅₀), the Hill slope (Hill) as well as themaximum (Max) and minimum (Min) signals in the assay.

$\left\lbrack {{hC}\; 3} \right\rbrack = {{Min} + \frac{{Max} - {Min}}{1 + \left( \frac{\lbrack{Alterase}\rbrack}{{EC}_{50}} \right)^{Hill}}}$

The cleavage of hC3 by modified MTSP-1 polypeptides with the sequenceset forth in SEQ ID NO: 35 was measured independently a total of 46times, using 9 different lots of the protease. The modified MTSP-1polypeptide with the sequence set forth in SEQ ID NO: 35 cleavedcomplement protein C3 with a lower EC₅₀ than the reference MTSP-1polypeptide set forth in SEQ ID NO: 4, which has an EC₅₀=13.9 nM (n=235;SD=4.1). The average EC₅₀ value for the modified MTSP-1 polypeptide withthe sequence set forth in SEQ ID NO: 35 was determined to be 6.9 nM(n=46, SD=2.6). C3 cleavage reactions were performed 1-12 times for allother modified MTSP-1 polypeptides listed in Table 15.

Table 15 sets forth exemplary modifications, and the EC₅₀ forpolypeptides, as set forth in the Sequence Listing, that contain thesemodifications. It is understood that the sequence listing sets forth theprotease domain, but that these same mutations can be included infull-length modified MTSP-1, and various forms thereof, andcatalytically active forms thereof. As set forth, all include thereplacement of the free cysteine C122S; the replacement reducesaggregation, and can be optional.

TABLE 15 hC3 Cleavage with Modified MTSP-1 Polypeptides SEQ IDChymotrypsin Numbering EC₅₀ NO.* MODIFICATIONS (nM) 4 C122S 13.9 23Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/ 14.4T98G/F99L/C122S/G151N/Q175L/Q192D 24Q38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/ 6.68T98P/F99L/C122S/G151H/Q175L/Q192E 32Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/ 2.87F99L/C122S/G151N/Q175L/Q192D 35Q38H/I41S/D60bT/F60eS/Y60gW/D96K/ins97aV/ 6.86F97G/T98P/F99L/C122S/G151H/Q175L/Q192D 36Q38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ 2.85ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D 37Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ 5.92ins97aA/T98P/F99L/C122S/G151N/Q175L/Q192D 38Q38H/I41S/D60bT/F60eS/Y60gW/F97G/ins97aV/ 169T98P/F99L/C122S/G151H/Q175L/Q192D 39Q38H/I41S/D60bT/F60eS/Y60gW/D96K/ins97aV/ 12T98P/F99L/C122S/G151H/Q175L/Q192D 40Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ 1.45ins97aV/T98P/F99L/C122S/G151H/Q175L 42 I41D/C122S/G151N/Q192T 85.2 43I41S/F99L/C122S/G151N/Q192V 34.4 44 I41E/F99L/C122S/G151N/Q192T 124 45I41D/Y59F/D96E/F99L/C122S/G151N/Q192T 54.9 46I41D/Y59F/C122S/G151N/Q192T 56.7 47I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 17.1T98P/F99L/C122S/G151H/Q175L/Q192D 48Q38H/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 215T98P/F99L/C122S/G151H/Q175L/Q192D 49Q38H/I41S/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/ 13.1F99L/C122S/G151H/Q175L/Q192D 50Q38H/I41S/D60bT/Y60gW/D96K/F97G/ins97aV/T98P/ 8.64F99L/C122S/G151H/Q175L/Q192D 51Q38H/I41S/D60bT/F60eS/D96K/F97G/ins97aV/T98P/ 22.2F99L/C122S/G151H/Q175L/Q192D 52Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/T98P/ 7.88F99L/C122S/G151H/Q175L/Q192D 53 Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/9.83 ins97aV/F99L/C122S/G151H/Q175L/Q192D 54Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ 18.3ins97aV/T98P/C122S/G151H/Q175L/Q192D 55Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ 5.25ins97aV/T98P/F99L/C122S/Q175L/Q192D 56Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ 19.9ins97aV/T98P/F99L/C122S/G151H/Q192D 57Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/ 108 Q192D 58I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/ 68.6 Q192D 59Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/ 22 Q175L/Q192D 77I41T/F97W/F99L/C122S/G151N/Q175M/Q192G/D217L 12.1 78I41G/F97L/F99L/C122S/Q175A/Q192T/D217V 7.75 79I41G/F97V/F99L/C122S/G151Q/Q175M/Q192A/D217L 9.74 80I41G/F97I/F99L/C122S/G151L/Q175M/Q192S/D217V 2.84 81I41G/F97S/F99L/C122S/G151N/Q175L/Q192G/D217I 6.84 154F97E/F99L/C122S/D217I/K224N 6.33 155 C122S/G193A 44.7 156 C122S/G193E119 157 D96_F97delinsWYY/T98P/F99L/C122S 22.6 158 F97D/F99L/C122S/Q192G78.1 159 H40R/I41H/F97D/F99L/C122S/Q192G 35.4 160 C122S/G151N/G193A 85.7161 H40R/I41H/C122S/G151N 59.6 162 H40R/I41H/F97D/C122S/G151N 110 163H40R/I41H/F97E/C122S 51.7 164 F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192E259 165 H40R/I41H/Y60gL/F97D/F99L/C122S/G151N/ 7.41 Q175M/D217I/K224S166 H40R/I41H/F97D/F99L/C122S/G151D/Q192G 1530 167H40R/I41H/F97D/F99L/Q192G 24.1 168H40R/I41H/Y60gH/F97D/F99L/C122S/G151N/ 19.8 Q175A/Q192H/D217I/K224R 169H40R/I41H/Y60gF/F97D/F99L/C122S/Q192G/ 38 D217M/K224R 170H40R/I41H/Y60gF/F97D/F99L/C122S/Q192G/ 25.9 D217R/K224A 171H40R/I41H/F97D/F99L/C122S/Q175L/Q192G/ 14.1 D217K/K224A 172H40R/I41H/F97D/F99L/C122S/Q175M/Q192G/ 2.76 D217V/K224Y 173H40R/I41H/F97D/F99L/C122S/Q175K/Q192G/ 19.4 D217I/K224H 174H40R/I41H/F97D/F99L/C122S/Q175M/Q192G/D217S 24.4 175H40R/I41H/Y60gF/F97D/F99L/C122S/Q175M/Q192G/ 9.92 D217W/K224R 176H40R/I41H/Y60gN/F97D/F99L/C122S/G151N/Q175K/ 187 Q192S/D217S/K224L 177H40R/I41H/Y60gH/F97D/F99L/C122S/Q175M/ 3.72 Q192G/D217I/K224L 178H40K/I41L/Y60gF/F97D/F99L/C122S/G151N/Q175R 237 179H40R/I41H/Y60gL/F97D/F99L/C122S/G151N 54.9 180H40K/I41M/Y60gG/F97D/F99L/C122S/G151N/ 509 Q175R/Q192R/D217V/K224S 181H40K/I41M/Y60gF/F97D/F99L/C122S/G151N/ 589 Q175L/Q192D 182H40R/I41H/F97D/C122S/G151N/Q175M/Q192A/ 470 D217S/K224R 183H40R/I41H/Y60gH/F97D/F99L/C122S/Q175M/ 7.94 Q192G/D217I/K224R 184H40R/I41H/F97D/F99L/C122S/G151D/Q175M/ 70.8 Q192G/D217V 185H40R/I41H/F97D/F99L/C122S/G151N/Q175M/ 563 Q192A/D217N/K224R 186H40R/I41H/F97D/F99L/C122S/G151N/Q175L/ 540 Q192A/D217N/K224R 187H40K/I41M/F97D/F99L/C122S/G151N/Q175M/ 9.74 Q192D/D217N/K224R 188H40K/I41M/F97D/F99L/C122S/G151N/Q175L/ 1290 Q192A/D217N/K224R 189H40R/I41H/F97D/F99L/C122S/Q175M/Q192D/ 9990 D217N/K224R 190H40R/I41H/F97D/F99L/C122S/Q175M/D217N/ 103 K224R 191H40K/I41M/F97D/F99L/C122S/Q175M/Q192D/ 9.64 D217N/K224R 192H40K/I41M/F97D/F99L/C122S/G151N/Q175M/ 1230 Q192A/D217N/K224R 193H40K/I41M/F97D/F99L/C122S/Q175M/D217N/ 144 K224R 194H40R/I41H/F97T/ins97aE/T98G/F99L/C122S/ 5860 Q175L/Q192E 195H40R/I41H/F97T/ins97aE/T98G/F99L/C122S/ 46 Q175L/Q192G 196H40R/I41H/F97E/ins97aE/T98G/F99L/C122S/ 82.4 Q175L/Q192G 197H40R/I41H/F97D/F99L/C122S/G151N/Q192H 179 198H40R/I41H/F97D/F99L/C122S/G151N/L153R 59.9 199H40R/I41H/F97D/C122S/G151N/L153R/V202M 103 200H40R/I41H/F97D/F99L/C122S/G151N/Q192H/P232S 262 201H40R/I41H/F97D/ins97aE/T98G/F99L/C122S/ 39.7 Q175L/Q192G 202H40R/I41H/F97D/C122S/G151N/L153R 126 203H40K/I41M/F99L/C122S/T150A/G151R/Q192G 110 204H40R/I41H/F97D/C122S/G133D/G151N 60.4 205 I41R/F99L/C122S/Q192G 53.8 206H40R/I41H/F99L/C122S/G151K/Q192G 1520 207I41R/ins97aE/F97T/T98G/F99L/C122S/G151E/ 126 Q175L/Q192E 208K86R/K110R/C122S/K134R/K157R/K224R/K239R 17.3 209H40R/I41H/K86R/F97D/K110R/C122S/K134R/ 304 G151N/K157R/K224R/K239R 210K86R/F97T/ins97aE/T98G/F99L/K110R/C122S/ 253K134R/K157R/Q175L/Q192E/K224R/K239R 211H40R/I41H/F97D/F99L/C122S/Q175R/Q192G/ 45.2 D217H/K224S 212H40R/I41H/F97D/F99L/C122S/Q192G/D217I/K224S 19.3 213H40R/I41H/F97D/F99L/C122S/Q192G/D217K/K224A 23.4 214H40R/I41H/F97D/F99L/C122S/Q175R/Q192G/ 31.2 D217E/K224R 215H40R/I41H/F97D/C122S/Q175R/Q192G/D217I/ 93.1 K224Q 216H40P/I41R/F99L/C122S/Q192G 62.4 217 H40P/I41R/F99L/C122S/G151K/Q192G96.1 218 H40R/I41H/F99L/C122S/G151E/Q192G 829 219I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/ 163 Q175L/Q192E 220I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/ 743 Q175T/Q192E 221I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/ 1620 Q175T/Q192D 222I41R/ins97aE/F97T/T98G/F99L/C122S/G151E/ 1770 Q175T/Q192D 223H40P/I41R/ins97aE/F97T/T98G/F99L/C122S/Q175L/ 442 Q192E 224H40P/I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/ 212 Q175L/Q192E 225I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/ 311 Q175L/Q192E 226I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/ 622 Q175L/Q192D 227I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/ 1410 Q175T/Q192E 228I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/ 9990 Q175T/Q192D 229I41R/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E 523 230H40P/I41R/F99L/C122S/G151E/Q192G 153 231I41R/F97T/ins97aE/T98G/F99L/C122S/G151E/ 363 Q175T/Q192E 232I41R/F97T/ins97aE/T98G/F99L/C122S/G151N/ 267 Q175S/Q192E 233I41R/F97T/ins97aE/T98G/F99L/C122S/G151N/ 1030 Q175I/Q192E 234H40R/I41H/Y60gF/F97D/F99L/C122S/Q175K/ 93.4 Q192G/D217R/K224Q 235H40R/I41H/F97D/F99L/C122S/Q175L/Q192G/ 14.9 D217Q/K224R 236H40R/I41H/F97D/F99L/C122S/G151N/Q192N/ 346 D217L/K224R 237H40R/I41H/F97D/F99L/C122S/G151N/Q192H/ 137 D217K/K224A 238ins97aV/F97D/T98P/F99L/C122S/Q192G 53.5 239 F97N/ins97aT/T98Y/F99N/C122S184 240 F97M/ins97aD/T98D/F99L/C122S/Q192T 151 241ins97aV/F97Q/T98P/F99L/C122S/Q175F/Q192D 248 242ins97aD/F97T/T98S/F99L/C122S/Q192E/D217Y/ 5000 K224R 243ins97aN/F97H/T98D/F99L/C122S/Q192E/D217Q/ 2750 K224S 244F97Q/ins97aT/T98M/C122S/Q192E/D217R/K224L 2450 245ins97aD/F97Q/T98G/F99L/C122S/Q175L/Q192E/ 5560 D217F/K224S 246ins97aD/F97G/T98N/F99L/C122S/Q192E/D217Y/ 9870 K224R 247ins97aE/F97Y/T98S/F99L/C122S/Q192T/D217Q/ 249 K224R 248ins97aG/F97N/T98D/F99L/C122S/Q192E/D217H/ 2790 K224A 249ins97aA/F97G/T98N/F99L/C122S/Q175M/Q192T/ 200 K224A 250I41R/ins97aE/F97T/T98G/F99L/C122S/G151N/ 2080 Q175S/Q192D 251I41R/F97T/ins97aE/T98G/F99L/C122S/G151N/ 8370 Q175I/Q192D 252I41R/F97T/ins97aE/T98G/F99L/C122S/G151D/ 2070 Q175I/Q192E 253I41R/ins97aE/F97T/T98G/F99L/C122S/G151D/ 1750 Q175I/Q192D 254S90T/D96A/ins97aE/F97T/T98G/F99L/C122S/ 280 Q175L/Q192D 255Y59F/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E 341 256ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E/ 383 Q209L 257Y59F/D96V/ins97aE/F97T/T98G/F99L/C122S/ 293 Q175L/Q192E 258D96V/ins97aE/F97T/T98G/F99L/C122S/Q175L/ 258 Q192D 259I41R/ins97aE/F97T/T98G/F99L/C122S/G151S/ 274 Q175L/Q192E 260E24K/ins97aE/F97T/T98G/F99L/C122S/A152S/ 291 Q175L/Q192D 261ins97aE/F97T/T98G/F99L/C122S/L153Q/Q175L/ 332 Q192D 262ins97aE/F97T/T98G/F99L/C122S/I136M/L155M/ 429 N170D/Q175L/Q192E 263I41R/ins97aE/F97T/T98G/F99L/A112V/C122S/ 288 Q175L/Q192E 264Y59F/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q192D 289 265Y59F/G60dS/R84H/ins97aE/F97T/T98G/F99L/C122S/ 102 Q175L/Q192E/V212I 266Y59F/ins97aE/F97T/T98G/F99L/C122S/L153Q/ 237 Q175L/Q192E 267I41R/Y59F/ins97aE/F97T/T98G/F99L/C122S/Q175L/ 288 Q192D 268I41R/Y59F/G60dS/R84H/ins97aE/F97T/T98G/F99L/ 143 C122S/Q175L/Q192E/V212I269 I41R/Y59F/ins97aE/F97T/T98G/F99L/C122S/L153Q/ 253 Q175L/Q192E 270I41R/F97W/F99L/C122S/G151N/Q192G 97.3 271F97D/ins97aV/T98P/F99L/C122S/G151N/Q192G 87 272I41D/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192E 157 273I41D/ins97aE/F97T/T98G/F99L/C122S/G151N/ 148 Q175L/Q192E 274Q38E/H40R/I41H/F97D/F99L/C122S/Q192G 30.9 275H40R/I41H/F97D/F99L/Q175R/Q192G/D217E/K224R 21.9 276I41R/ins97aV/F97D/T98P/F99L/C122S/G151N/Q192G 232 277ins97aV/F97D/T98P/F99L/C122S/Q175L/Q192E 158 278I41R/ins97aV/F97D/T98P/F99L/C122S/G151N/ 187 Q175L/Q192E 279Q38E/H40R/I41H/D60bE/F97D/F99L/C122S/Q192G 28.1 280Q38E/H40R/I41H/D60bN/F97D/F99L/C122S/Q192G 39.5 281Q38E/H40R/I41H/D60bK/F97D/F99L/C122S/Q175L/ 15.3 Q192G 282Q38E/H40R/I41H/D60bN/F60eT/F97D/F99L/C122S/ 9.42 Q175L/Q192G 283Q38R/I41S/D60bH/F60eV/F97T/ins97aE/T98G/F99L/ 35.2 C122S/Q175L/Q192E 284Q38G/H40R/I41H/D60bK/F97D/F99L/C122S/Q175L/ 20.6 Q192G 285I41D/ins97aE/F97T/T98G/F99L/C122S/G151N/ 103 Q175L/Q192E/Q209L 286Q38G/H40R/I41H/D60bN/F97D/F99L/C122S/Q175L/ 0.867 Q192G 287Q38R/I41S/D60bH/F60eV/ins97aE/F97T/T98G/F99L/ 22.5C122S/G151N/Q175L/Q192E 288H40R/I41H/F97D/ins97aV/T98P/F99L/C122S/Q175R/ 65.8 Q192G/D217E/K224R 289Q38H/I41S/D60bA/F60eV/Y60gF/F97T/ins97aE/ 7.46T98G/F99L/C122S/Q175L/Q192T 290Q38E/I41S/D60bH/F60eI/F97T/ins97aE/T98G/F99L/ 10.4 C122S/Q175L/Q192V 291Q38R/I41S/D60bH/F60eI/ins97aE/F97T/T98G/F99L/ 27.3 C122S/Q175L/Q192E 292Q38E/I41S/D60bV/F60eK/F97T/ins97aE/T98G/F99L/ 20.4 C122S/Q175L/Q192I 293Q38R/I41E/ins97aE/F97T/T98G/F99L/C122S/Q175L/ 63.3 Q192T 294Q38H/I41A/D60bV/F60eR/Y60gW/ins97aE/F97T/ 2.37T98G/F99L/C122S/Q175L/Q192D 295Q38H/I41A/D60bA/F60eR/F97T/ins97aE/T98G/F99L/ 12 C122S/Q175L/Q192E 296F97D/ins97aV/T98P/F99L/C122S/G151N/Q175L/ 194 Q192E 297ins97aV/F97D/T98P/F99L/C122S/G151N/Q175L/ 38.5 Q192G 298Q38G/H40R/I41H/D60bN/F60eT/F97D/F99L/C122S/ 11.9 Q175L/Q192G 299Q38G/H40R/I41H/D60bK/F60eT/F97D/F99L/C122S/ 21.3 Q175L/Q192G 300Q38E/H40R/I41H/D60bK/F60eT/F97D/F99L/C122S/ 16.6 Q175L/Q192G 301Q38H/I41S/D60bA/F60eV/Y60gF/ins97aE/F97T/ 14.2T98G/F99L/C122S/G151N/Q175L/Q192T 302Q38E/I41S/D60bH/F60eI/ins97aE/F97T/T98G/F99L/ 11.5C122S/G151N/Q175L/Q192V 303Q38R/I41S/D60bH/F60eI/ins97aE/F97T/T98G/F99L/ 32.9C122S/G151N/Q175L/Q192E 304Q38E/I41S/D60bV/F60eK/ins97aE/F97T/T98G/F99L/ 19.1C122S/G151N/Q175L/Q192I 305Q38R/I41E/ins97aE/F97T/T98G/F99L/C122S/G151N/ 105 Q175L/Q192T 306Q38H/I41A/D60bV/F60eR/Y60gW/ins97aE/F97T/ 7.74T98G/F99L/C122S/G151N/Q175L/Q192D 307Q38H/I41A/D60bA/F60eR/ins97aE/F97T/T98G/F99L/ 14.2C122S/G151N/Q175L/Q192E 308Q38H/I41S/D60bT/F60eS/ins97aV/F97D/T98P/F99L/ 29.8C122S/G151H/Q175L/Q192E 309 Q38H/I41S/D60bV/F60eQ/Y60gF/ins97aE/F97T/5.95 T98G/F99L/C122S/Q175L/Q192I 310Q38H/I41A/D60bV/F60eI/ins97aE/F97T/T98G/F99L/ 7.64 C122S/Q175L/Q192E 311Q38H/I41A/D60bV/F60eT/Y60gW/ins97aE/F97T/ 1.39T98G/F99L/C122S/Q175L/Q192E 312 Q38H/I41A/F60eA/Y60gW/ins97aE/F97T/T98G/5.93 F99L/C122S/Q175L/Q192E 313Q38H/I41A/D60bE/F60eH/Y60gW/ins97aE/F97T/ 5.38T98G/F99L/C122S/Q175L/Q192D 314Q38H/I41A/D60bT/F60eK/Y60gW/ins97aE/F97T/ 3.82T98G/F99L/C122S/Q175L/Q192D 315Q38H/I41A/D60bT/F60eH/Y60gW/ins97aE/F97T/ 2.28T98G/F99L/C122S/Q175L/Q192D 316Q38H/I41S/D60bS/F60eR/Y60gW/F97T/ins97aE/ 8.35T98G/F99L/C122S/Q175L/Q192E 317Q38H/I41S/D60bT/F60eS/F97D/ins97aV/T98P/F99L/ 45.4C122S/G151H/Q175L/Q192D 318Q38R/I41T/ins97aE/F97T/T98G/F99L/C122S/G151N/ 30.6 Q175L/Q192G 319Q38S/I41S/F60eR/F97T/ins97aE/T98G/F99L/C122S/ 6.26 G151N/Q175L/Q192S 320Q38H/I41T/D60bV/F60eQ/F97T/ins97aE/T98G/ 4.93 F99L/C122S/Q175L/Q192G 321Q38G/H40R/I41H/D60bH/F60eK/F97T/ins97aE/ 27.7T98G/F99L/C122S/Q175L/V183A/Q192G 322Q38H/I41A/F97T/ins97aE/T98G/F99L/C122S/ 3.9 Q175L/Q192G 323Q38L/I41T/D60bR/F60eL/Y60gM/F97T/ins97aE/ 23.7T98G/F99L/C122S/G151N/Q175L/Q192G 324Q38F/I41S/D60bF/F60eR/Y60gF/F97T/ins97aE/ 1.96T98G/F99L/C122S/G151N/Q175L/Q192V 325Q38V/I41S/D60bT/F60eT/F97D/ins97aV/T98P/F99L/ 18.5C122S/G151N/Q175H/Q192S 326 Q38W/I41A/ins97aV/F97D/T98P/F99L/C122S/ 68G151T/Q175S/Q192D 327 Q38T/I41S/D60bV/F60eR/ins97aV/F97D/T98P/F99L/ 15.7C122S/G151N/Q175R/Q192V 328Q38H/I41S/D60bT/F60eS/ins97aV/F97D/T98P/F99L/ 67.8C122S/G151H/Q175A/Q192D 329Q38H/I41S/D60bT/F60eT/F97D/ins97aV/T98P/F99L/ 5.08C122S/G151N/Q175L/Q192V 330Q38Y/I41A/D60bL/F60eQ/ins97aV/F97D/T98P/F99L/ 1.53C122S/G151N/Q175M/Q192A 331Q38L/I41T/D60bA/F60eL/ins97aV/F97D/T98P/F99L/ 20.2C122S/G151H/Q175M/Q192T 332Q38R/I41S/D60bY/F60eD/ins97aV/F97D/T98P/F99L/ 3.22C122S/G151N/Q175M/Q192A 333Q38W/I41S/D60bG/F60eI/F97D/ins97aV/T98P/F99L/ 43.1C122S/G151N/Q175A/Q192D 334 Q38T/I41S/D60bG/F60eM/ins97aV/F97D/T98P/28.8 F99L/C122S/G151N/Q175S/Q192S 335I41T/D60bW/F60eH/F97D/ins97aV/T98P/F99L/ 3.1 C122S/G151N/Q175L/Q192G 336Q38D/I41S/D60bT/F60eR/ins97aV/F97D/T98P/F99L/ 48.1C122S/G151K/Q175S/Q192V 337Q38H/I41S/D60bF/F60eV/F97D/ins97aV/T98P/F99L/ 1.57C122S/G151N/Q175L/Q192A 338 Q38L/I41A/D60bH/F60eT/ins97aV/F97D/T98P/5.73 F99L/C122S/G151Q/Q175A/Q192G 339Q38H/I41A/D60bE/F60eH/Y60gW/F97T/ins97aE/ 8.28T98G/F99L/C122S/G151N/Q175L/Q192D 340Q38H/I41A/D60bV/F60eI/Y60gW/F97T/ins97aE/ 2.91T98G/F99L/C122S/G151N/Q175L/Q192D 341Q38E/I41S/D60bV/F60eK/Y60gW/F97T/ins97aE/ 12.1T98G/F99L/C122S/G151N/Q175L/Q192D 342Q38H/I41S/D60bT/F60eS/Y60gW/ins97aV/F97D/ 12.6T98P/F99L/C122S/G151H/Q175L/Q192D 343Q38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/ 22.6T98P/F99L/C122S/G151H/Q175A/Q192D 344Q38H/I41A/D60bV/F60eR/ins97aE/F97T/T98G/ 6.06F99L/C122S/G151N/Q175L/Q192D 345Q38H/I41A/D60bT/F60eH/Y60gW/F97T/ins97aE/ 6.95T98G/F99L/C122S/G151N/Q175L/Q192D 346D60bY/F97T/ins97aE/T98G/F99L/C122S/Q175L/ 4.1 Q192G 347I41T/D60bY/F97T/ins97aE/T98G/F99L/C122S/ 3.88 Q175L/Q192G 348Q38E/I41S/D60bT/F60eR/F97T/ins97aE/T98G/F99L/ 32.7 C122S/Q175L/Q192V 349Q38H/I41A/D60bK/F60eK/Y60gW/F97T/ins97aE/ 22.3T98G/F99L/C122S/Q175L/Q192D 350 Q38H/I41S/D60bA/F60eV/ins97aE/F97T/T98G/16.9 F99L/C122S/Q175L/Q192E/Q209L 351Q38H/I41A/D60bT/F60eR/F97T/ins97aE/T98G/ 6.54 F99L/C122S/Q175L/Q192V 352Q38H2I/I41S/F97T/ins97aE/T98G/F99L/C122S/ 43 Q175L/Q192V 353Q38F/I41A/D60bT/F60eG/Y60gW/ins97aE/F97T/ 1.25T98G/F99L/C122S/Q175L/Q192E 354 Q38H/I41A/F60eH/Y60gW/ins97aE/F97T/T98G/3.09 F99L/C122S/Q175L/Q192A 355Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/ 2.71T98G/F99L/C122S/Q175L/Q192A 356Q38H/I41A/D60bV/F60eA/Y60gW/F97T/ins97aE/ 1.57T98G/F99L/C122S/Q175L/Q192V 357Q38E/I41V/D60bF/F60eK/Y60gF/F97T/ins97aE/ 14.2T98G/F99L/C122S/Q175L/Q192G 358 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623I41G/Y60gW/F99L/C122S/Q175R/Q192S 6.95 624I41A/Y60gW/ins97aE/F99L/C122S/Q175M/Q192T 8.9 625I41T/ins97aA/F99Y/C122S/Q175L/Q192A 28 626I41A/ins97aY/F99L/C122S/Q175R/Q192H 14.8 627I41S/ins97aT/F99L/C122S/Q175R/Q192H 27.5 628I41S/Y60gW/ins97aN/F99L/C122S/Q175R/Q192T 12 629Q38H/I41S/D96S/ins97aK/C122S/G151N/Q192A 16.7 630Q38H/I41A/D96A/ins97aA/C122S/G151D/Q192T 9.25 631Q38H/I41S/D96Q/ins97aT/C122S/G151N/Q192A 17.9 632Q38H/I41T/D96M/ins97aA/C122S/G151D 10.8 633 Q38Y/I41A/D96I/ins97aQ/C122S4.26 634 Q38H/I41S/D96K/ins97aT/C122S/G151K/Q192A 19.2 635Q38W/I41S/D96R/ins97aA/C122S/G151N/Q192A 9.5 636Q38H/I41A/D96R/ins97aQ/C122S 7.6 637 Q38F/I41V/D96Q/ins97aT/C122S/G151D9.89 638 L33M/Q38F/I41S/D96A/ins97aW/C122S/G151N/ 10.6 Q192S 639Q38H/I41S/D96V/ins97aA/C122S/G151N/Q192A 16 640Q38H/I41T/D96K/ins97aL/C122S/G151N/Q192A 44.7 641Q38H/I41S/D96Q/ins97aA/C122S/Q192T 10.2 642Q38W/I41V/D96R/ins97aA/C122S/G151N 4.49 643Q38Y/I41T/D96M/ins97aS/C122S/G151N 12.5 644Q38H/I41S/D96K/ins97aS/C122S/G151P/Q192S 25.6 645Q38H/I41S/D96G/ins97aG/C122S/G151N/Q192A 34.6 646Q38H/I41S/D96K/ins97aD/C122S/G151N/Q192S 26.8 647I41S/D96E/ins97aG/C122S/G151Q/Q192A 44.4 648I41S/Y59F/ins97aV/C122S/G187D/Q192V/D217V 8.35 649A35V/I41S/Y59F/C122S/Q192D/D217V 39.8 650I41S/F93L/ins97aV/C122S/Q192V/D217V 9.82 651I41S/S90P/ins97aV/C122S/Y146E/Q192N/D217V 6.47 652I41S/S90T/ins97aV/C122S/Q192N/D217V 13.4 653I41S/S90T/ins97aV/C122S/Q192V/D217V 10.5 654I41S/Y59F/ins97aV/C122S/Q192G 25.6 655I41S/Y59F/F97S/ins97aV/S116Y/C122S/Q192G/ 8.22 D217V 656I41S/ins97aV/C122S/Q192G/Q209L 33.4 657Q38H/I41S/ins97aV/A112V/C122S/Q192A/Q209L 10.9 658I41S/ins97aV/C122S/Q192V/D217V 11 659 I41S/Y59F/ins97aV/C122S/Q192A 16.6660 I41A/F97G/ins97aM/T98L/C122S 40.1 661 I41G/F97E/F99L/C122S/Q192A30.1 662 I41S/F97V/ins97aV/T98P/C122S 30.9 663I41S/T98S/F99L/C122S/Q192A 17.4 664 I41S/F97Q/F99L/C122S/Q192S 53.5 665I41G/F97L/F99L/C122S/Q192S 41.4 666 I41S/F97G/ins97aA/T98P/C122S/Q192A57.2 667 I41A/F97G/ins97aV/T98E/C122S 18.1 668 I41A/F97S/ins97aA/C122S19.8 669 I41A/F97W/T98S/F99L/C122S/Q192A 27 670I41L/N95D/D96T/F97W/F99L/C122S/Q192A 62.6 671I41T/Y60gL/N95D/D96F/F97S/F99L/C122S/Q175S/ 247 Q192A 672I41A/Y60gW/N95D/D96F/F97G/F99L/C122S/Q175H/ 17.5 Q192A 673I41A/Y60gW/F99L/C122S/Q175T/Q192A 17.6 674Q38M/I41T/D96M/ins97aH/C122S/G151E 19.1 675Q38H/I41T/D96R/ins97aG/C122S/G151S 54.1 676I41S/D60bY/ins97aV/T98N/C122S/Q192H 8.1 677I41S/Y59F/D60bY/ins97aV/C122S/Q192G 5.24 678I41S/D60bY/ins97aV/A112V/C122S/Q192G/Q209L 4.76 679A35T/I41S/Y59F/ins97aV/C122S/Y146F/V183A/ 18.4 Q192G/R235H 680I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/ 195 Q175H/Q192D 681I41S/ins97aV/C122S/N164D/Q192G/R235H 46.4 682I41S/Y59F/ins97aV/C122S/Q192G/N223D 34.1 683I41S/ins97aV/C122S/N164D/Q192G/R235L 49.7 684I41S/Y59F/F97Y/ins97aV/C122S/Q192G 29.1 685I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q192V 32.3 686I41S/F99L/C122S/G151N/Q175M/Q192G/D217V 3.19 687I41S/F97L/F99L/C122S/G151N/Q192G/D217V 11.9 688I41S/F97S/F99L/C122S/G151N/Q175L/Q192A/D217L 11.2 689I41G/F97R/F99L/C122S/G151N/Q175L/Q192S/D217V 4.18 690I41T/F97L/F99L/C122S/G151N/Q175S/Q192S/D217W 24.2 691I41D/F97T/F99M/C122S/Q192V/D217M 63.8 *SEQ ID of the protease domaincontaining the replacements; it is understood that these replacementscan be included in full-length MTSP-1 and in other variants, includingcatalytically active fragments thereof.Among these of interest are those with an EC₅₀ for hC3 cleavage of lessthan 10, such as, but are not limited to, for example:

EC₅₀ SEQ ID (nM) Mutation string NO. 0.866ins97aA/F97G/T98L/C122S/Q175M/Q192A/D217I/K224R 368 0.867Q38G/H40R/I41H/D60bN/F97D/F99L/C122S/Q175L/Q192G 286 1.08Q38Y/I41S/D60bT/F60eR/Y60gW/D96M/F97N/T98G/F99L/C122S/G151N/Q1 491 75L1.11 Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G 432151H/Q175L/Q192E 1.17Q38H/I41S/D60bT/F60eS/Y60gW/D96F/F97D/ins97aE/T98S/F99L/C122S/G15 4251H/Q175L/Q192A 1.25Q38F/I41A/D60bT/F60eG/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q 353192E 1.31Q38H/I41S/D60bT/F60eS/Y60gW/ins97aV/F97D/T98P/F99L/C122S/G151H/Q 447175L 1.39Q38H/I41A/D60bV/F60eT/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q 311192E 1.43Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aA/T98P/F99L/C122S/G1 49651H/Q175L/Q192D 1.45Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G1  4051H/Q175L 1.51Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G 413151N/Q175L/Q192E 1.53Q38Y/I41A/D60bL/F60eQ/ins97aV/F97D/T98P/F99L/C122S/G151N/Q175M/Q 330192A 1.57Q38H/I41S/D60bF/F60eV/F97D/ins97aV/T98P/F99L/C122S/G151N/Q175L/Q1 33792A 1.57Q38H/I41A/D60bV/F60eA/Y60gW/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q 356192V 1.57Q38H/I41A/D60bV/F60eR/Y60gW/D96I/ins97aN/F97Y/T98G/F99L/C122S/G1 42851H/Q175L/Q192D 1.59Q38H/I41S/D60bT/F60eS/Y60gW/D96L/ins97aG/F97D/T98N/F99L/C122S/G1 42651H/Q175L/Q192E 1.83Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G 434151H/Q175L/Q192D 1.96Q38F/I41S/D60bF/F60eR/Y60gF/F97T/ins97aE/T98G/F99L/C122S/G151N/Q1 32475L/Q192V 1.97Q38H/I41S/D60bY/D96Y/ins97aV/F97D/T98P/F99L/L106M/C122S/I136M/Q1 40792G/Q209L/D217T 2.07Q38H/I41A/D60bV/F60eR/Y60gW/D96S/ins97aR/F97A/T98S/F99L/C122S/G1 41251N/Q175L/Q192T 2.23Q38H/I41A/D60bV/F60eR/Y60gW/D96I/ins97aN/F97Y/T98G/F99L/C122S/G1 40851N/Q175L/Q192D 2.28Q38H/I41A/D60bT/F60eH/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q 315192D 2.37Q38H/I41A/D60bV/F60eR/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q 294192D 2.42 ins97aY/F97G/T98V/C122S/Q175M/Q192S/D217V 372 2.43Q38H/I41S/D60bS/ins97aV/F97D/T98P/F99L/M117L/C122S/I136T/Q192G/D2 38617I 2.52 Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C122S/G151H/Q17429 5L/Q192D 2.54Q38H/I41A/D60bV/F60eR/Y60gW/D96P/ins97aN/F97W/T98G/F99L/C122S/G 433151N/Q175L/Q192D 2.71Q38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/C122S/Q175L/Q 355192A 2.76 H40R/I41H/F97D/F99L/C122S/Q175M/Q192G/D217V/K224Y 172 2.84I41G/F971/F99L/C122S/G151L/Q175M/Q192S/D217V  80 2.85Q38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/T98P/F99L/C122S/G1  3651H/Q175L/Q192D 2.87Q38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C122S/G151N/Q17  325L/Q192D 2.88Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/T15 4950S/G151H/Q175L/Q192D/Q209L 2.91Q38H/I41A/D60bV/F60eI/Y60gW/F97T/ins97aE/T98G/F99L/C122S/G151N/Q 340175L/Q192D 3.04Q38H/I41S/D60bT/F60eS/Y60gW/D96I/F97N/T98G/F99L/C122S/G151N/Q175 436L/Q192D 3.06Q38H/I41A/D60bW/ins97aV/F97D/T98P/F99L/C122S/I136M/Q192G/D217N 388 3.07Q38H/I41S/D60bF/F60eT/ins97aV/F97D/T98P/F99L/C122S/H143Q/G151N/Q1 41975L/Q192G 3.08Q38Y/I41S/D60bT/Y60gW/D96M/F97N/T98G/F99L/C122S/G151N/Q175L/Q1 484 92D3.09 Q38H/I41A/F60eH/Y60gW/ins97aE/F97T/T98G/F99L/C122S/Q175L/Q192A 3543.1 I41T/D60bW/F60eH/F97D/ins97aV/T98P/F99L/C122S/G151N/Q175L/Q192G 3353.13 Q38H/I41S/D60bT/F60eS/Y60gW/D96F/F97Y/ins97aD/T98G/F99L/C122S/G1472 51H/Q175L/Q192D 3.14Q38H/I41A/D60bV/F60eR/Y60gW/D96F/F97S/ins97aH/T98G/F99L/C122S/G1 46951N/Q175L/Q192G 3.19Q38H/I41S/D60bT/F60eS/Y60gW/ins97aV/T98P/F99L/C122S/G151H/Q175L/ 441Q192E 3.19 I41S/F99L/C122S/G151N/Q175M/Q192G/D217V 686 3.22Q38R/I41S/D60bY/F60eD/ins97aV/F97D/T98P/F99L/C122S/G151N/Q175M/Q 332192A 3.27 Q38Y/I41S/D60bT/F60eR/Y60gW/D96M/T98G/F99L/C122S/G151N/Q175L/Q487 192D 3.72 H40R/I41H/Y60gH/F97D/F99L/C122S/Q175M/Q192G/D2171/K224L177 3.72 Q38H/I41A/D60bV/F60eR/Y60gW/F97D/F99L/C122S/G151H/Q175L/Q192D435 3.75 Q38Y/I41S/D60bT/F60eR/Y60gW/D96M/F97N/T98G/F99L/C122S/Q175L/Q1492 92D 3.76Q38H/I41A/D60bV/F60eR/Y60gW/D96F/F97Y/ins97aN/T98G/F99M/C122S/G 470151N/Q175L/Q192G 4.53Q38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97N/ins97aE/T98S/F99L/C122S/G15 4731H/Q175L/Q192D 5.92Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F99L/C122S/G1  3751N/Q175L/Q192D 6.86Q38H/I41S/D60bT/F60eS/Y60gW/D96K/ins97aV/F97G/T98P/F99L/C122S/G1  3551H/Q175L/Q192D

Example 3

Confirmation of cleavage at the targeted Q H A R↓A S H L site in C3

Two independent analytical methods were used to characterize thecleavage site(s) of C3 by the MTSP-1 protease domain variant thatcontains the modificationsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D:(a) direct analysis of cleavage products by MALDI-MS with identificationof released peptides in the 1-40 kDa range and (b) LC-tandem MS of alabeled proteolytic digest with sequence analysis of product peptidesthat contain a neo-N-terminus. These experiments were performed asfollows:

(a) C3 (1 mg/ml in PBS buffer) was incubated with MTSP-1 proteasevariant at enzyme to substrate ratios of 1:50 for a total of one hour.Samples (50 μl) were removed from these reactions at 0, 5, 10, 20, 40,and 60 minutes, and the cleavage reaction was terminated in each sampleby addition of 1 μl 1% TFA and flash freezing in dry ice. Samples werethen reduced with 25 mM TCEP (1 hr at 37° C.), then desalted via C18solid phase extraction (Agilent Omix). The resulting cleavage productswere analyzed directly by MALDI-MS (ABI 4700). At each time point after0 minutes a fragment of MW 8289±1 Da was observed, indicating cleavageat the arginine in the QHAR|ASHG site in C3. No additional C3 cleavagesites were observed in these reactions.

(b) The C3/protease variant mixture (10 μl) was denatured in 6Mguanidine, then cysteine side chains were reduced (30 mM TCEP) andalkylated (iodoacetamide). The ε-amino group of lysine side chains wasblocked by treatment with O-methylisourea (3 μl OMU, 8 μl 1 M NaOH, 15min at 65° C.; quench with 2 μl 1:1 TFA-water, followed by SPE cleanup)and then peptide amino termini were labeled with SulfoNHS-SS-biotin (50mM HEPES, 250 uM biotin reagent, 30 min at RT). After proteolyticdigestion with either trypsin or Glu-C, the biotin-labeled peptides werecaptured by avidin beads. Cleavage of the biotin label was achieved withreduction (TCEP), giving a neo-N-terminal peptide fraction with aN-thioacyl label. This peptide fraction was analyzed by LC-tandem MS(Thermo LTQ-XL); the major C3-related component identified was thepeptide (N-thioacyl)-ASHGLAR, indicating cleavage at the QHAR|ASHG sitein C3.

-   -   Q H A R↓A S H L 737-744    -   P4 P3 P1↓P1′ P4′.

Example 4 Ex Vivo Pharmacodynamic (PD) Analysis of MTSP-1 Variants inCynomolgus Monkey Plasma

The cleavage of C3 in anti-coagulated EDTA-treated cynomolgus monkeyplasma by the wild type and modified MTSP-1 polypeptides was measured asdescribed below. A C3 ELISA was used to measure the effective dose(ED₅₀) of wild type and modified MTSP-1 polypeptides required to cleave50% of the C3 in the “test,” anti-coagulated plasma. The cleavagereactions contained 80% plasma, and each protease was assayed at 9different concentrations in addition to a zero protease control. Thehighest concentration of wild type MTSP-1 used in this reaction was 6 μMand the next eight concentrations were prepared by sequential dilutionsby a factor of 1.5. The highest concentration used for MTSP-1 variantproteins was 150 nM and, as with the wild type protein, the next eightconcentrations were prepared by sequential dilutions by a factor of 1.5.Following addition of test protease, the reaction was incubated for 10minutes at 37° C. The reaction was then rapidly quenched by addition ofthe protease inhibitor 10 μM EGR-CMK (Glu-Gly-Arg-chloromethyl ketone)and the quenched samples were placed at room temperature for 30 minutesbefore performing the C3 ELISA. Cleavage reactions were diluted 1:2700in BSA-PBST and uncleaved C3 was “captured” with Goat anti huC3 (A213CompTech) (adsorbed to a microtiter plate) and “detected” by 0.5 μg/mlMAB anti-huC3a (Abcam ab11872) in 1% BSA-PBST. The ELISA was then“developed” using Goat anti Mouse HRP conjugate and the WesternBrightSirius Western Blotting Detection Kit following the manufacturer'sdirections.

TABLE 16 hC3 cleavage with modified MTSP-1 polypeptides in Cynomolgusmonkey plasma SEQ ID ED₅₀ 80% cynomolgus Chymotrypsin numbering NO.*plasma (nM) Wild-type MTSP-1 protease domain with C122S 4 2800I41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192 21 2200 EQ38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99 22 101L/C122S/G151N/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99 23 195L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99 24 76L/C122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99 25 152L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99 26 136L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99 27 118L/C122S/G151N/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99 28 133L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97Y/ins97aN/T98 29 37G/F99L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98 30 85P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96P/F97W/ins97aN/T9 31 738G/F99L/C122S/G151N/Q175L/Q192EQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/ 32 58C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97E/ins97aV/T98 33 133G/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96L/F97D/ins97aG/T98 34 70N/F99L/C122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98 35 92P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/T98 36 103P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98 37 37P/F99L/C122S/G151N/Q175L/Q192D *SEQ ID of the protease domain containingthe replacements

Example 5 Ex Vivo Stability of Modified MTSP-1 Polypeptides inCynomolgus Monkey Vitreous Humor

The ex vivo stability of modified MTSP-1 polypeptides was assessed inpurchased cynomolgus monkey vitreous humor or Phosphate Buffered Saline(PBS) negative control. Modified MTSP-1 polypeptides that exhibitstability in vitreous humor can be used for treatment of AMD.

80% Cynomolgus vitreous humor (obtained from BioChemed; Catalog Nos.BC7615-V1, BC60815-V1, BC33115-V6) in buffer containing 50 mM Tris pH8.0, 50 mM NaCl, and 0.01% Tween-20 or PBS control was incubated withmodified MTSP-1 polypeptides at a final concentration of 0.1 μM. Themixture was incubated at 37° C. for 7 days. The residual proteaseactivity was assayed with 100 μM fluorogenic substrate AGR-ACC(7-amino-4-carbamoylmethyl-coumarin) in 50 mM Tris, pH 8.0, 50 mM NaCl,0.01% Tween-20 and the results were assessed at excitationwavelength=380 nm and emission wavelength=460 nm. The results show thatthe modified MTSP-1 polypeptides with the sequences set forth in SEQ IDNOs: 35, 38-40, and 47-56 exhibit comparable residual activity (i.e.,stability) after incubation in cynomolgus plasma and PBS. The resultsare set forth in Table 17 below.

TABLE 17 Stability of MTSP-1 polypeptides in vitreous humor SEQ IDActivity (%) on Day 7 Chymotrypsin numbering NO.* vitreous PBS Wild-typeMTSP-1 protease domain with C122S 4 59 63Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 35 92 94T98P/F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97G/ins97aV/T98P/ 38 73 91F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/ins97aV/T98P/ 39 77 85F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 40 18 23T98P/F99L/C122S/G151H/Q175LI41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/ 47 86 87F99L/C122S/G151H/Q175L/Q192DQ38H/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/ 48 76 78F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F 49 87 8199L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/Y60gW/D96K/F97G/ins97aV/T98P/ 50 85 91F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/D96K/F97G/ins97aV/T98P/F 51 85 9399L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/T98P/F9 52 19 349L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 53 66 82F99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 54 94 98T98P/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 55 74 87T98P/F99L/C122S/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/ 56 90 94T98P/F99L/C122S/G151H/Q192D *SEQ ID of a protease domain containing thereplacements

The ex vivo stability of the modified MTSP-1 polypeptides in purchasedCynomolgus monkey vitreous humor after 7 and 28 days was assessed asabove. The results show that the modified MTSP-1 polypeptides providedherein are relatively stable for at least 7 days in vitreous humor. Theresults are set forth in Table 18 below.

TABLE 18 SEQ ID Activity (%) at 37° C. Chymotrypsin numbering NO.* Day 7Day 28 Wild-type MTSP-1 protease domain with C122S 4 67 47Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97a 35 91 68V/T98P/F99L/C122S/G151H/Q175L/Q192D I41E/F99L/C122S/G151N/Q192T 41 10091 I41D/C122S/G151N/Q192T 42 71 35 I41S/F99L/C122S/G151N/Q192V 43 79 NDI41E/F99L/C122S/G151N/Q192T 44 91 90I41D/Y59F/D96E/F99L/C122S/G151N/Q192T 45 85 NDI41D/Y59F/C122S/G151N/Q192T 46 88 86Q38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q 57 95 80 192DI41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q175L/ 58 96 75 Q192DQ38H/I41S/D96K/F97G/ins97aV/T98P/F99L/C122S/Q 59 92 63 175L/Q192D *SEQID of the protease domain containing the replacements

Example 6 Ex Vivo Pharmacodynamic Activity in Human Plasma

Serial dilutions of modified MTSP-1 polypeptides (or buffer) were addedto human plasma (that contains ˜8 μM endogenous C3) to create reactionmixtures that contained 6000, 4000, 2667, 1778, 1185, 790, 527, 351, 234or 0 nM concentrations of each variant polypeptide and 80% human plasma.Similar reaction mixtures were prepared for wild type MTSP with the wildtype MTSP present at concentrations of 150, 100, 67, 44, 30, 20, 13, 9,6 or 0 nM. These reaction mixtures were incubated for 1 hour at 37° C.and quenched with 10 μM EGR-CMK. Each reaction mixture was diluted 1:15,625 in PBST buffer containing 1% BSA and the residual, uncleaved C3concentration in the mixture was “detected” using mAb anti-huC3a (Abcamab11872) and the assay signal was “developed” using HRP conjugate Goatanti Mouse-HRP (JIR 115-035-003) and the WesternBright Sirius WesternBlotting Detection Kit. These data were used to calculate theconcentration of each MTSP polypeptide required to cleave 50% of the C3present in the plasma during the 1 hour incubation (i.e., the ED₅₀).

The results are shown in Table 19 below, which sets forth the ED₅₀ (nM)of hemolysis in 80% human plasma by the reference MTSP-1 protease domaincomprising the WT-MTSP-1 protease domain with the C122S replacement, andthe modified MTSP-1 polypeptides. As shown in Table 19, the ED₅₀ for thereference MTSP-1 polypeptide in 80% human plasma is 3500 nM whereasexemplary MTSP-1 polypeptides have increased ability to cleavecomplement as indicated by a lower ED₅₀ (e.g., between 24 nM and 835nM). For example, the modified MTSP-1 polypeptide with the sequence setforth in SEQ ID NO: 35, which contain the replacementsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,was approximately 140-fold more potent than the reference MTSP-1protease domain with the C122S replacement.

TABLE 19 ED₅₀ 80% human SEQ ID plasma Chymotrypsin numbering NO.* (60min, nM) Wild-type MTSP-1 protease domain with C122S 4 3500I41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192E 21 835Q38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99L/C 22 52122S/G151N/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/C 23 65122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C1 24 5022S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C1 25 5022S/G151H/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/C 26 38122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C1 27 4122S/G151N/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99L/C 28 32122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97Y/ins97aN/T98G/F 29 2799L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F 30 3499L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96P/F97W/ins97aN/T98G/ 31 47F99L/C122S/G151N/Q175L/Q192EQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C12 32 242S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97E/ins97aV/T98G/F 33 3899L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96L/F97D/ins97aG/T98N/F 34 3999L/C122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F 35 2599L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/T98P/F 36 2799L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F 37 4499L/C122S/G151N/Q175L/Q192D *SEQ ID of the protease domain containingthe replacements

Example 7 Cleavage of Proteinase-Activated Receptor 2 (PAR-2)

A. Activity of Mutants in a Proteinase-Activated Receptor 2 Cell-BasedAssay

Wild type MTSP-1 is an efficient activator of PAR-2, G-protein-coupledreceptor expressed in vascular endothelial cells and a variety ofepithelial cells that is involved in inflammatory diseases such asarthritis, lung inflammation (asthma), inflammatory bowel disease,sepsis, and pain disorders. Reduced PAR-2 activity by an anti-C3 variantMTSP-1 polypeptide, therefore, increases C3 selectivity for thatengineered protease. Consequently, the modified MTSP-1 polypeptides weretested for their activity (i.e., ability to activate) on theproteinase-activated receptor 2 (PAR-2) in a cell based assay(Millipore). The ED₅₀ (nM) of PAR-2 cleavage by the proteases weremeasured by plotting the fraction cleavage vs. protease concentration ona 4 parameter logistic curve fit (SoftMax Pro software, MolecularDevices, CA). A catalytically inactive version of MTSP-1 was provided asa negative control. Decreased PAR-2 activity in this assay (i.e., ahigher ED₅₀ for PAR-2 cleavage versus wild type MTSP-1) for a variantMTSP-1 polypeptide indicates that the variant protein displays morerestricted specificity than wild type MTSP-1. Increased specificityversus PAR-2 indicates that the variant also can exhibit decreasednon-specific cleavage of other proteins compared with wild type MTSP-1.The results of these assays (see Table 20 below) demonstrate that thepolypeptides display significantly reduced PAR-2 activity compared withwild type MTSP-1.

Another assay used to mimic the proteolytic activation of PAR-2 in vivomeasures the activity of variant MTSP-1 polypeptides on a quenchedfluorescence peptidic substrate containing the peptide sequence SKGR↓SL,the P4-P2′ sequence (Ser 33 to Leu 38) of the activation cleavage sitein PAR-2. Cleavage of R↓S site in this peptidic substrate produces afluorescence signal allowing measurement of the reaction rate with afluorescence plate reader. Reduced activity towards the SKGR/SL peptidicsubstrate compared with that of wild type MTSP-1 indicates that thevariant MTSP-1 polypeptide possesses enhanced substrate specificity.

Exemplary results are shown in Table 20, below. All tested modifiedMTSP-1 polypeptides exhibited at least a 30-fold increased ED₅₀ anddecreased k_(cat)/K_(m) compared to wild-type MTSP-1 protease domainwith the C122S replacement set forth in SEQ ID NO: 4. The data indicatethat the modified MTSP-1 polypeptides selected for cleavage of C3 havesignificantly reduced activity for a native substrate.

TABLE 20 PAR-2 Cell SEQ SKGR/SL Based ID k_(cat)/K_(m) Assay ED₅₀Chymotrypsin numbering NO.* (M⁻¹s⁻¹) (nM) Wild-type MTSP-1 proteasedomain with C122S 4 200000 1.4I41R/F97T/Ins97aE/T98G/F99L/C122S/G151N/Q175L/Q192E 21 <100 2600Q38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99L/ 22 925 690C122S/G151N/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/ 23 245 1900C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C 24 959 1800122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C 25 1730 640122S/G151H/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97aE/T98G/F99L/ 26 742 560C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97aV/T98P/F99L/C 27 1730 n.d.122S/G151N/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97aE/T98G/F99L/ 28 1360 430C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97Y/ins97aN/T98G/ 29 2320 450F99L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F 30 4030 42099L/C122S/G151H/Q175L/Q192DQ38H/I41A/D60bV/F60eR/Y60gW/D96P/F97W/ins97aN/T98G/ 31 1620 n.d.F99L/C122S/G151N/Q175L/Q 192EQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97N/T98G/F99L/C1 32 3560 15022S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96Y/F97E/ins97aV/T98G/F 33 865 n.d.99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96L/F97D/ins97aG/T98N/F 34 1300 n.d.99L/C122S/G151H/Q175L/Q192EQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F 35 6210 n.d.99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96V/F97G/ins97aV/T98P/F 36 4990 n.d.99L/C122S/G151H/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97D/ins97aA/T98P/F 37 2060 n.d.99L/C122S/G151N/Q175L/Q192D *SEQ ID of the protease domain containingthe replacements

Example 8 Pharmacokinetic and Pharmacodynamic Activity in CynomolgusMonkey Vitreous Humor In Vivo

The in vivo pharmacodynamic activity in vitreous humor (cynomolgusmonkey model) of the modified MTSP-1 polypeptide set forth in SEQ ID NO:35, which is the protease domain that contains the replacementsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,was assessed. The ability to cleave and inactivate C3 in vitreous humorare indicative of a candidate for treatment of AMD.

Twelve naive cynomolgus monkeys were assigned to a single treatmentgroup. Study animals were intravitreally administered a single dose of125 μg of modified MTSP-1 polypeptide in one eye. The isolated proteasedomain whose sequence is set forth in SEQ ID NO: 35, which has amolecular weight of approximately 25 kDa, was administered. The righteye received the test article and the left eye was injected with vehiclecontrol. Four animals were sacrificed at each of the following timepoints: 24 hours post-dose, day 2 and on day 6. Vitreous humor sampleswere collected from both the right and left eyes and analyzed formodified MTSP-1 polypeptide stability and level of C3 after treatmentwith modified MTSP-1 polypeptide or vehicle control; C3 and modifiedMTSP-1 polypeptide concentrations were determined by ELISA as detailedabove.

The concentration of the modified MTSP-1 polypeptide present in vitreoushumor samples obtained 24 hours post-dose and on day 2, day 6, day 7,and day 28 was measured by ELISA. Proteolytic activity of the MTSP-1polypeptides (and other serine proteases) in the vitreous samples wasquenched by the addition of EGR-CMK (Haematologic Technologies,EGRCK-01) to a final concentration of 10 μM, and the mixture was allowedto stand for 30 minutes at ambient temperature before performing theELISA.

The half-life of the modified MTSP-1 polypeptide of SEQ ID NO: 35 wasdetermined to be approximately 1.7 days, which corresponds toapproximately 5 days in a human system (Deng et al. (2011) MAbs 3(1):61-66). In vivo recovery (i.e., the peak level of modified MSTP-1polypeptide detected divided by the dose of the modified MTSP-1polypeptide) of the modified MTSP-1 polypeptide set forth in SEQ ID NO:35 was calculated by ELISA from the observed maximum level of themodified MTSP-1 polypeptide set forth in SEQ ID NO: 35. The theoreticalpredicted value for 100% in vivo recovery was 2.5 μM. The measured invivo recovery of the MTSP-1 protease domain (SEQ ID NO: 35) containingthe replacementsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192Dwas calculated to be approximately 59% of the predicted value, orapproximately 1.5 μM. There was substantial variation between twoseparate experiments (measurement 1=100% and measurement 2=18%recovery), indicating either poor intravitreal injections orinappropriate sample handling, storage, or dilution, and thereforeincomplete local delivery of active anti-C3 protease into the vitreous,in the second experiment.

C3 levels in vitreous humor were measured by ELISA. C3 levels invehicle-injected negative control eye ranged between 0.4 nM-50 nM (2samples from vehicle-injected eyes differed significantly from the other10, likely due to blood contamination during harvesting of the vitreouswith a needle). The baseline level of C3 prior to MSTP-1 administrationwas approximately 2.2 nM. C3 was undetectable in the variant-treated eye1, 2, 6, and 7 days after the single injection of the anti-C3 MTSPpolypeptide. 28 days after the single injection, the C3 concentrationwas approximately 1.4 nM in the eye treated with modified MTSP-1polypeptide set forth in SEQ ID NO: 35, which is approximately 64% ofthe baseline level before treatment.

Results show that the modified MTSP-1 polypeptide catalyticallyeliminates C3. It had a half-life of 1.7 days, as assessed by ELISA andenzyme assays, and suppresses vitreal complement for at least, or longerthan, 7 days. Doses of up to 1 mg/eye were well tolerated. PK/PDmodeling indicates a suppression of C3 for 3 months or more in humans.

Example 9 Exemplary Mutations at Positions in MTSP-1

Exemplary positions and mutations of MTSP-1 polypeptides, including thefull-length, precursor and protease domains and catalytically activeportions thereof, are set forth in Table 21 below.

TABLE 21 Exemplary mutations at positions in MTSP-1 Chymotrypsin MatureSEQ ID Exemplary Conservative to numbering numbering WT NO. 35 mutationsMutations 38 637 Q H H N, Q 41 640 I S S, R, A, E, D T, K, Q 59 658 Y FM, L, Y 60b 661 D T T, V S, I, L 60e 664 F S S, R, K T, Q, E 60g 666 Y WW Y 96 705 D K K, V, Y, L, I, R, Q, E, W, F P 97 706 F G G, T, D, E, P,S, Q, H, F N, Y, W Ins97a V V, E, A, G, N I, L, S, P, Q, H, D 98 707 T PP, G, N Q, H 99 708 F L L LV 151 759 G H H, N Q 175 783 Q L L LV 192 802Q D D, E Q

The replacements are in any form of MTSP-1, including the proteasedomain (SEQ ID NO: 2 or 4) and the full length (SEQ ID NO: 1 or 3). Thereplacements can be combined, including as exemplified herein, includingup to as many as 15-18 or more replacements.

Example 10 In Vivo Safety, Tolerability, and Toxicity Studies(Cynomolgus Monkey) of MTSP-1 Variants Following Intravitreal Injection

Safety and tolerability of Modified MTSP-1 polypeptides followingintravitreal injection were assessed in vivo in cynomolgus monkeys.Three naive cynomolgus monkeys were assigned to each of three treatmentgroups. Study animals were intravitreally administered either 12.5 μg,37.5 μg or 125 μg per eye, of each modified MTSP-1 polypeptide. Theright eye received the test polypeptide and the left eye was injectedwith vehicle control. Animals were clinically observed (i.e., foodconsumption) and ophthalmic examinations were conducted. Ophthalmicexamination included slit-lamp biomicroscopy and indirect ophthalmoscopeobservations, followed by color fundus photography or optical coherencetomography (OCT) prior to dosing (T=0) and on days 2, 8 and 15post-dosing. All observations continued for up to 4 weeks or untilresolution.

The no-observed-adverse-effect-level (NOAEL) was assessed for allanimals. The NOAEL for animals administered a modified MTSP-1polypeptide with the sequence set forth in SEQ ID NO:42 was ≥37.5 μg(equivalent to ≥125 μg/eye in man). No adverse effects were noted foranimals administered a modified MTSP-1 polypeptide with the sequence setforth in SEQ ID NO:35; therefore, the NOAEL for animals administered amodified MTSP-1 polypeptide set forth in SEQ ID NO:35 was ≥125 μg(equivalent to ≥375 μg/eye in man).

Example 11 Pharmacodynamic Activity, Safety/Toxicity, and TherapeuticIndex Following Intravenous Injection of MTSP-1 Polypeptides

The NOAEL following intravenous injection was assessed in a cynomolgusmonkey model. The highest non-toxic dose for cynomolgus monkeysadministered a modified MTSP-1 polypeptide set forth in SEQ ID NO:35 was≥4 mg/kg. The ED₅₀ for inactivation of circulating C3 was also measured.The C3 activity (i.e., ED₅₀ for inactivation of C3) for animalsadministered a modified MTSP-1 polypeptide set forth in SEQ ID NO:35 was0.07 mg/kg, which was significantly lower than that of WT MTSP-1. The“therapeutic index” (T.I.) of an anti-C3 MTSP-1 variant polypeptide wasdefined as the ratio of the NOAEL and the ED₅₀ for inactivation of C3 invivo. The results are set forth in Table 22, below:

TABLE 22 C3 destruction SEQ ID ED₅₀ NOAEL Single Chymotrypsin numberingNO. (mg/kg) (mg/kg) Bolus T.I. Q38H/I41A/D60bV/F60eR/Y60gW/F97T/ins97a22 0.2 ≥0 NA E/T98G/F99L/C122S/G151N/Q175L/Q192DQ38H/I41A/D60bT/F60eK/Y60gW/F97T/ins97a 23 0.2 ≥4 ~20E/T98G/F99L/C122S/G151N/Q175L/Q192DQ38H/I41S/D60bT/F60eS/Y60gW/F97D/ins97a 24 0.1 ≥2 ~20V/T98P/F99L/C122S/G151H/Q175L/Q192EQ38H/I41A/D60bV/F60eR/Y60gW/D96I/F97Y/ 29 0.06 ≥1 ~17ins97aN/T98G/F99L/C122S/G151N/Q175L/Q19 2DQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ 35 0.07 ≥4 >57ins97aV/T98P/F99L/C122S/G151H/Q175L/Q19 2D

Example 12 Demonstration that the Modified MTSP-1 Polypeptides Cleave C3and Inhibit Complement Activation

A. Demonstration of the Complement Inhibitory Effect of an Anti-C3Protease (Sequence ID NO:35) in Human Plasma

1. In Vitro Inhibition of Complement Activation in Human Plasma by theMTSP-1 Variant Polypeptide of Sequence ID NO:35

Studies were performed to assess the anti-complement activity of MTSP-1polypeptide modified to cleave C3 in human plasma. The test article inthese experiments, exemplary of the modified polypeptides described inthis application, was the modified MTSP-1 polypeptide of SEQ ID NO:35,which is the protease domain that contains the replacementsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D.The test article (referred to as test article #1, below), andexperimental controls were exposed to the test system, pooled citratedplasma.

The extent of complement inhibition was assessed by measuring inhibitionof the standard classical pathway hemolytic assay (CH50). The testcitrated plasma was exposed to the test article prior to complementanalysis. The extent of complement inhibition is the decrease inhemolytic lysis in the CH50 and C3 Function testing compared with thatobserved with control (i.e., untreated) citrated plasma. The C3 Functiontesting allows for analysis of the specific inhibition of the C3component of the complement cascade.

2. Test Systems

Testing was performed with human citrated plasma pool only (NETS, poolsof 3-5 normal individuals).

3. Route of Administration

The test article was added to the test system at a ratio of one part tonine parts (1:9, V:V). This ratio maintains appropriate concentration ofthe test system so there is sufficient concentration of the complementcontrol proteins. The testing was performed by mixing 450 μL of testsystem with 50 μL of prepared test article in a 1.5 mL polypropylenemicrocentrifuge snap cap tube. The mixture was prepared on ice and thenvortexed to mix. After all experimental mixtures (positive and negativecontrols and multiple concentrations of test article) were prepared,they were transferred to a 37° C.±1° C. water bath and incubated for 1hour±10 minutes. After the mixtures were incubated, if necessary, anyparticulates were removed by centrifugation, and all samples aliquotedon ice and immediately frozen at −70° C. or below.

4. Test Articles Descriptions

Test Article Storage Concentration Other Considerations Test Article≤−65° C. 4 mg/mL 100 μL is provided #1 151.6 μM Buffer is PBS

5. Control Articles

Positive Control #2 Source Storage Concentration Other ConsiderationsHAGG (Activator of the Exsera Biolabs −70° C. 10 mg/mL None ClassicalPathway)* 1 *HAGG—heat activated gamma globulin

Negative Control #1 Source Storage Concentration Other ConsiderationsSaline Exsera 4° C. 0.9% Saline (154 mmol NaCl) None Biolabs

6. Control and Test Article Preparation

Five concentrations of test article were used with the highest level at2000 nM. Lower levels were generated with a five-fold serial dilution.

Dilution Table:

10× Amount of Concentration in test Concentration Stock to Which Amount(nM) (μM) add (μL) stock of saline Total Volume(μL) 2000 20 30 151.6 200230 400 4 40 20 160 200 80 0.8 40 4 160 200 16 0.16 40 0.8 160 200 3.20.032 40 0.16 160 200

All dilutions to be made with saline unless otherwise indicated

7. Experimental Design & Data

The testing included five concentrations of test article.

Test or Control Final Concentration Article Tube Label (nM unlessindicated) Test Article #1 1-A 2000 1-B 400 1-C 80 1-D 16 1-E 3.2 Saline11-S NA Neat (no additive) 12-N NA Zymosan 13-Z 1 mg/mL HAGG 14-H 1mg/mL

8. Results

Test or Control Tube Final Concentration Data % Article Label (nM unlessindicated) U/mL Inhibition Test Article #1 1-A 2000 5.56 92% 1-B 40010.84 84% 1-C 80 41.45 40% 1-D 16 68.97  1% 1-E 3.2 72.98 −5% Saline SNA 69.32 HAGG H 1 mg/mL 8.16

These data demonstrate that an anti-C3 MTSP-1 polypeptide (Sequence IDNO:35) effectivity inhibits complement activity in human plasma with 40%inhibition observed at a “dose” of 80 nM anti-C3 MTSP-1 variant(Sequence ID NO:35) and near complete inhibition (i.e., 94%) observed atthe highest dose of the MTSP-1 polypeptide used in the studies.

B. Demonstration that Cleavage at the QHAR/ASHL Site Inactivates HumanC3

To confirm that the complement inhibition by Test Article 1 (MTSP-1polypeptide of Sequence ID NO:35) demonstrated above is mediated bycleavage of C3 at the QHAR/ASHL site, the experiment described below wasperformed.

1. Experimental Design Summary

The complement function assay was performed with serum deficient of C3(purchased from Complement Technologies; catalog No. A314). C3 (alsopurchased from Complement Technologies; catalog No. A113) was added backto the serum with and without pre-incubation with a compositioncontaining the test article #1 (the modified MTPS-1 polypeptide of SEQID NO:35). The degree to which the pre-incubation with Test Article #1inhibits complement function reflects the level of inhibition of C3.

2. Description of Purified C3 Reagent

Normal concentration of C3 in human serum is −1 mg/mL. The concentrationof the C3 from Complement Technologies is 1.1 mg/ml. The C3 was added tothe depleted C3 serum at a 1/50 dilution.

3. Test Article Description

Test Article Storage Concentration Other Considerations Test Article≤−65 °C. 4 mg/mL 100 μL is provided #1 151.6 μM Buffer is PBS

4. Controls

Positive Control #2 Source Concentration HAGG (Activator of the Exsera10 mg/mL Classical Pathway)* Biolabs *HAGG—heat activated gamma globulin

Negative Control #1 Source Concentration Saline Exsera Biolabs 0.9%Saline (154 mmol NaCl)

5. Control and Test Article Preparation

Dilution Table:

Concentration 10x Amount of Total in test Concentration Stock to addWhich Amount Volume (nM) (μM) (μL) stock of saline (μL) NT 20 30 151.6200 230 200 2 20 20 180 200 40 0.4 40 2 160 200 8 0.08 40 0.4 160 200All dilutions made with saline unless otherwise indicated.

6. Experimental Design: Test Conditions 1 and 2

Test Condition 1:

Components (Tube 1 and Tube 2) were incubated separately for 2 hours at37° C. (±2° C.). Tube 1 contained 100 μL C3, and tube 2 contained 25 μlof the test article (TA, MTSP-1 polypeptide or saline). Samples werefrozen at −80° C. or below until testing in C3H50 with depleted serum.Each tube was incubated at 37° C. for 2 hours. 90 μl C3 from tube 1 wascombined with 10 μl (TA, polypeptide or saline) from tube 2, then mixedand frozen immediately at 80° C.; no prior cleavage of C3 by the TAbefore addition to C3H50 at 1/50 dilution.

Test Condition 2:

Components were mixed in tube 3 (90 μL C3 and 10 μl TA, light vortex),and incubated for 2 hours (pre-cleavage of C3 at the QHAR site (residues737-740 of SEQ ID NO:9) at 37° C. (±2° C.), frozen immediately, andadded to C3H50 at 1/50.

Final Concen- tration in Test Test or Condition Control Tube PurifiedTest (nM unless Expected Article Label C3 (Y/N) Conditions indicated)Outcome Saline Only S N Test NA Zero C3H50 Condition #1 Saline SC Y TestNA Full C3H50 Condition #2 Test 1-A Y Test 200 Full C3H50 Article #1 1-BY Condition #1  40 or mildly 1-C Y  8 inhibited Test 2-A Y Test 200 LowC3H50 Article #1 2-B Y Condition #2  40 inverse to TA 2-C Y  8concentration Neat N N Test NA Condition #1 Zymosan Z N Test 1 mg/mLCondition #1 HAGG H N Test 1 mg/mL Condition #1

7. Readout of Complement Activation or Inhibition

Modified C3H50 Hemolytic Function. C3H50 is a measure of the functionalactivity but with specific emphasis on C3, as it requires the C3 addedexogenously. The serum was made deficient in C3. The preparedexperimental conditions (conditions 1 and 2) are added to the deficientserum at a 1/50 dilution for the testing. The in-test incubation withthe red blood cells was performed at 22° C. for 45 minutes.

8. Results

The results show that inhibition of complement activation, as assessedby hemolytic activity, is mediated by cleavage of C3.

Final Concentration Test or Purified in Test Hemolytic Control Tube C3Test Condition Activity Article Label (Y/N) Conditions* (nM) (U/mL)Saline Only S N Test NA 82.43 Condition #1 Saline SC Y Test NA 375.81Condition #2 Test 1-A Y Test 200 437.77 Article #1 1-B Y Condition #1 40 594.13 1-C Y  8 470.03 Test 2-A Y Test 200 73.67 Article #1 2-B YCondition #2  40 97.89 2-C Y  8 295.35 *Test condition #1: Incubatecomponents separately for 2 hours at 37° C. (±2° C.). Then freeze at−80° C. or below until testing in C3H50 (C3 hemolytic activity) withdepleted serum (i.e., no pre-cleavage of serum C3). *Test condition #2:Mix components together with light vortexing and incubate for 2 hours at37° C. (±2° C.) (i.e., pre-cleavage of serum C3 by the MTSP-1polypeptide (Sequence ID NO: 35).

These data demonstrate that preincubation of C3 with the MTSP-1 variantof Sequence ID NO:35 (that cleaves C3 at the QHAR/ASHL site)substantially inhibits complement activation in human serum. A 1 hourpreincubation of human serum with the MTSP-1 variant reduces thehemolytic activity of the serum by approximately 80%.

Example 13 Site Specific Mutagenesis of an Exemplary MTSP-1 Variant toEstablish Structure-Activity Relationships for Individual Mutations inMTSP-1

To assess the effect of each replacement, insertion or deletion, sitespecific mutagenesis was used to create 14 variants of the MTSP-1variant that contains the modificationsQ38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D(starting variant). Each of the 14 variants contained a single mutation(compared with the starting variant) in which each single mutatedresidue in the starting variant (except C122S) was “reverted” (one ineach variant) to the corresponding amino acid present in wild typeMTSP-1. These variants are shown in FIG. 2 with reference to the WTMTSP-1 protease domain set forth in SEQ ID NO:4. Cells with bold bordersindicate that the modified MTSP-1 polypeptides at the indicated residuecontain the same amino acid as the wild-type MTSP-1 polypeptide setforth in SEQ ID NO:4. Cells without bold borders indicate that themodified MTSP-1 polypeptides contain the same amino acid as the modifiedMTSP-1 polypeptide set forth in SEQ ID NO:35.

The anti-C3 activity of each selected variant was assessed by measuringthe ED₅₀ for C3 cleavage as described above, and the stability of eachvariant was assessed by measuring the residual enzymatic activity, usingthe fluorogenic substrate AGR-ACC, after incubation for 7 days in eitherbuffer [Phosphate Buffered Saline (PBS)] or 80% cynomolgus monkeyvitreous humor as shown below. These data demonstrate that approximately50% of the “starting variant” polypeptides exhibit greater activityagainst C3 in vitreous humor than the reference wild type MTSP-1protease domain, whose sequence is set forth in SEQ ID NO:4. Inaddition, 12/14 of the modified MTSP-1 polypeptides are more stable invitreous humor compared with the reference wild-type protease domain ofSEQ ID NO:4, with some showing more stability than others. Therapeuticcandidates for ocular indications such as AMD, including those in thetable below, are variants that exhibit high C3 cleavage activity andhigh stability in vitreous humor.

The C3 activity (i.e., ED₅₀) of the modified MTSP-1 polypeptides wasmeasured in vitro as described above. The stability of the MTSP-1polypeptides after incubation for 7 days in either cynomolgus monkeyvitreous humor or Phosphate Buffered Saline (PBS) was measured with anactivity assay using the fluorogenic substrate AGR-ACC.

The data in the examples and above indicate that the modified MTSP-1polypeptides cleave human C3 efficiently and maintain 59-94% of thisactivity after incubation for 7 days in vitreous humor.

For example, the modified MTSP-1 polypeptides cleave human C3 betweenresidues 740 and 741 (SEQ ID NO:9) to thereby inactivate C3:

-   -   Residue No.    -   Q H A R↓A S H L 737-744    -   P4 P3 P1↓P1′ P4′.        Functional consequences of the modified MTSP-1 polypeptides,        such as the modified MTSP-1 polypeptides that contain the        mutations:    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/G151H/Q175L/Q192D        or    -   Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D,        where C122S is included to reduce aggregation (see, e.g., SEQ ID        NO:35, which sets forth a protease domain of a modified MTSP-1        polypeptides that contains these mutations) are as follows:    -   Q38H—This mutation increases C3 activity by approximately        3.7-fold.    -   I41S—This mutation increases anti-C3 activity by approximately        44.6-fold.    -   D60bT—This mutation increases anti-C3 activity by approximately        2.8-fold.    -   F60eS—This mutation increases anti-C3 activity by approximately        1.9-fold.    -   Y60gW—This mutation increases the enzyme's substrate specificity        and increases anti-C3 activity by approximately 4.8-fold.    -   D96K—This mutation increases anti-C3 activity by approximately        2.6-fold.    -   F97G—This mutation increases anti-C3 activity by approximately        4.3-fold and increases substrate specificity.    -   Insert 97aV: This mutation increases the modified MTSP-1        polypeptide substrate specificity, increases the modified MTSP-1        polypeptides' stability following 1 week incubation at 37° C. by        about 4.8-fold, and increases anti-C3 activity by approximately        1.7-fold.    -   T98P: This mutation increases the enzyme's stability following 1        week incubation at 37° C. by 1.4-fold and increases anti-C3        activity by approximately 2.2-fold.    -   F99L: This mutation increases anti-C3 activity by approximately        3.9-fold.    -   G151H: This mutation increases anti-C3 activity by approximately        1.2-fold.    -   Q175L: This mutation increases anti-C3 activity by approximately        4.3-fold.    -   Q192D: This mutation increases stability in vitreous humor        following 1 week incubation at 37° C. by 5.1-fold.        Among the polypeptides, those containing the mutations        Q38H/I41S/D60bT/F60eS/Y60gW/D96K/F97G/ins97aV/T98P/F99L/C122S/G151H/Q175L/Q192D        and I41D/C122S/G151N/Q192T are for use for treating DGF and/or        AMD.

All residues in the MTSP-1 polypeptides are referenced by chymotrypsinnumbering. Unmodified MTSP-1 polypeptides include those of SEQ ID NOs.:1-4, WT full-length MTSP-1, WT protease domain MTSP-1, WT mature MTSP-1,full-length MTSP-1 with C122S, protease domain MTSP-1 with C122S, matureMTSP-1 with C122S, respectively, where numbering is by chymotrypsinnumbering. All modified MTSP-1 polypeptides can include the replacementC122S in place of C122C.

* * *

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed:
 1. A modified membrane type serine protease (MTSP-1)polypeptide, comprising: an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, wherein said aminoacid sequence comprises, with reference to numbering in SEQ ID NO:1, thefollowing modifications: Q637H, I640S, D661T, F664S, Y666W, D705K,F706G, InsV, T707P, F708L, G759H, Q783L and Q802D (equivalent to Q38H,I41S, D60bT, F60eS, Y60gW, D96K, F97G, Ins97aV, T98P, F99L, G151H,Q175L, and Q192D, respectively, by chymotrypsin numbering); and whereinmodifications comprise insertions, deletions and/or replacements in theunmodified MTSP-1 polypeptide.
 2. The modified MTSP-1 polypeptide ofclaim 1, wherein the modified MTSP-1 polypeptide has at least 80%sequence identity to the polypeptide of SEQ ID NO:
 2. 3. The modifiedMTSP-1 polypeptide of claim 1, wherein the modified MTSP-1 polypeptidehas at least 90% sequence identity to the polypeptide of SEQ ID NO: 2.4. The modified MTSP-1 polypeptide of claim 1, wherein the unmodifiedMTSP-1 polypeptide consists of the amino acid sequence set forth in SEQID NO:2.
 5. The modified MTSP-1 polypeptide of claim 1 that isPEGylated.
 6. The modified MTSP-1 polypeptide of claim 4 that isPEGylated.
 7. The modified MTSP-1 polypeptide of claim 5, comprising aPEG that has a molecular weight from about 3 kD to about 50 kD.
 8. Themodified MTSP-1 polypeptide of claim 6, comprising a PEG that has amolecular weight from about 3 kD to about 50 kD.
 9. The modified MTSP-1polypeptide of claim 5, comprising a PEG that has a molecular weight of40 kD.
 10. The modified MTSP-1 polypeptide of claim 6, comprising a PEGthat has a molecular weight of 40 kD.
 11. The modified MTSP-1polypeptide of claim 7, comprising a branched PEG.
 12. The modifiedMTSP-1 polypeptide of claim 8, comprising a branched PEG.
 13. Themodified MTSP-1 polypeptide of claim 5, wherein the PEG is linked via acysteine residue in the modified MTSP-1 polypeptide.
 14. The modifiedMTSP-1 polypeptide of claim 6, wherein the PEG is linked via a cysteineresidue in the modified MTSP-1 polypeptide.
 15. The modified MTSP-1polypeptide of claim 12, wherein the PEG is linked via a cysteineresidue in the modified MTSP-1 polypeptide.
 16. The modified MTSP-1polypeptide of claim 15, wherein the modified MTSP-1 polypeptide cleavesa target site in C3 that inactivates C3.
 17. The modified MTSP-1polypeptide of claim 16, wherein the cleavage is between residues 740and 741 of SEQ ID NO:9, whereby cleavage occurs at Q H A R↓A S H L. 18.A pharmaceutical composition comprising the modified MTSP-1 polypeptideof claim 1 and a physiologically acceptable excipient.
 19. Apharmaceutical composition comprising the modified MTSP-1 polypeptide ofclaim 15 and a physiologically acceptable excipient.
 20. A method oftreating a disease or condition mediated by or associated with C3complement activation, comprising administering the pharmaceuticalcomposition of claim
 18. 21. A method of treating a disease or conditionmediated by or associated with C3 complement activation, comprisingadministering the pharmaceutical composition of claim
 19. 22. The methodof claim 20, wherein the disease or condition is macular degeneration ordiabetic retinopathy.
 23. The method of claim 21, wherein the disease orcondition is macular degeneration or diabetic retinopathy.
 24. Themethod of claim 22, wherein the disease or condition is age-relatedmacular degeneration (AMD).
 25. The method of claim 23, wherein thedisease or condition is age-related macular degeneration (AMD).
 26. Amodified MTSP-1 polypeptide comprising the amino acid sequence set forthin SEQ ID NO:35, except that the residue at position 731, with referenceto numbering in SEQ ID NO:1, is cysteine (C) (equivalent to the residueat position 122, by chymotrypsin numbering).
 27. The modified MTSP-1polypeptide of claim 26 that is PEGylated.
 28. The modified MTSP-1polypeptide of claim 27, comprising a PEG that has a molecular weightfrom about 3 kD to about 50 kD.
 29. The modified MTSP-1 polypeptide ofclaim 27, comprising a PEG that has a molecular weight of 40 kD.
 30. Themodified MTSP-1 polypeptide of claim 28, comprising a branched PEG. 31.The modified MTSP-1 polypeptide of claim 27, wherein the PEG is linkedvia a cysteine residue in the modified MTSP-1 polypeptide.
 32. Themodified MTSP-1 polypeptide of claim 29, comprising a PEG-maleimidelinked via a cysteine residue in the modified MTSP-1 polypeptide. 33.The modified MTSP-1 polypeptide of claim 30, comprising a PEG-maleimidelinked via a cysteine residue in the modified MTSP-1 polypeptide. 34.The modified MTSP-1 polypeptide of claim 31, comprising a PEG-maleimidelinked via a cysteine residue in the modified MTSP-1 polypeptide. 35.The modified MTSP-1 polypeptide of claim 34, wherein the modified MTSP-1polypeptide cleaves a target site in C3 that inactivates C3.
 36. Themodified MTSP-1 polypeptide of claim 35, wherein the cleavage is betweenresidues 740 and 741 of SEQ ID NO:9, whereby cleavage occurs at Q H AR↓A S H L.
 37. A pharmaceutical composition comprising the modifiedMTSP-1 polypeptide of claim 26 and a physiologically acceptableexcipient.
 38. A pharmaceutical composition comprising the modifiedMTSP-1 polypeptide of claim 34 and a physiologically acceptableexcipient.
 39. A method of treating a disease or condition mediated byor associated with C3 complement activation, comprising administeringthe pharmaceutical composition of claim
 37. 40. A method of treating adisease or condition mediated by or associated with C3 complementactivation, comprising administering the pharmaceutical composition ofclaim
 38. 41. The method of claim 39, wherein the disease or conditionis macular degeneration or diabetic retinopathy.
 42. The method of claim40, wherein the disease or condition is macular degeneration or diabeticretinopathy.
 43. The method of claim 41, wherein the disease orcondition is age-related macular degeneration (AMD).
 44. The method ofclaim 42, wherein the disease or condition is age-related maculardegeneration (AMD).
 45. The modified MTSP-1 polypeptide of claim 5,comprising a PEG-maleimide linked via a cysteine residue in the modifiedMTSP-1 polypeptide.
 46. The modified MTSP-1 polypeptide of claim 9,comprising a PEG-maleimide linked via a cysteine residue in the modifiedMTSP-1 polypeptide.
 47. The modified MTSP-1 polypeptide of claim 27,comprising a branched PEG.
 48. The modified MTSP-1 polypeptide of claim29, comprising a branched PEG.
 49. The modified MTSP-1 polypeptide ofclaim 27, comprising a PEG-maleimide linked via a cysteine residue inthe modified MTSP-1 polypeptide.
 50. The modified MTSP-1 polypeptide ofclaim 28, comprising a PEG-maleimide linked via a cysteine residue inthe modified MTSP-1 polypeptide.
 51. The modified MTSP-1 polypeptide ofclaim 47, comprising a PEG-maleimide linked via a cysteine residue inthe modified MTSP-1 polypeptide.
 52. The modified MTSP-1 polypeptide ofclaim 48, comprising a PEG-maleimide linked via a cysteine residue inthe modified MTSP-1 polypeptide.
 53. The modified MTSP-1 polypeptide ofclaim 45, comprising a branched PEG.
 54. The modified MTSP-1 polypeptideof claim 46, comprising a branched PEG.
 55. The modified MTSP-1polypeptide of claim 13, comprising a PEG-maleimide linked via acysteine residue in the modified MTSP-1 polypeptide.
 56. The modifiedMTSP-1 polypeptide of claim 14, comprising a PEG-maleimide linked via acysteine residue in the modified MTSP-1 polypeptide.